Cytokine receptor zalpha11

ABSTRACT

Novel polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods are disclosed for zalpha11, a novel cytokine receptor. The polypeptides may be used within methods for detecting ligands that stimulate the proliferation and/or development of hematopoietic, lymphoid and myeloid cells in vitro and in vivo. Ligand-binding receptor polypeptides can also be used to block ligand activity in vitro and in vivo. The polynucleotides encoding zalphall, are located on chromosome 16, and can be used to identify a region of the genome associated with human disease states. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to Provisional Application60/100,896, filed on Sep. 23, 1998. This application is also related toProvisional Application 60/123,546, filed on Mar. 3, 1999; andProvisional Application 60/142,574, filed on Jul. 6, 1999. Under 35U.S.C. § 119(e)(1), this application claims benefit of said ProvisionalApplications.

BACKGROUND OF THE INVENTION

[0002] Proliferation and differentiation of cells of multicellularorganisms are controlled by hormones and polypeptide growth factors.These diffusable molecules allow cells to communicate with each otherand act in concert to form cells and organs, and to repair damagedtissue. Examples of hormones and growth factors include the steroidhormones (e.g. estrogen, testosterone), parathyroid hormone, folliclestimulating hormone, the interleukins, platelet derived growth factor(PDGF), epidermal growth factor (EGF), granulocyte-macrophage colonystimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

[0003] Hormones and growth factors influence cellular metabolism bybinding to receptors. Receptors may be integral membrane proteins thatare linked to signaling pathways within the cell, such as secondmessenger systems. Other classes of receptors are soluble molecules,such as the transcription factors. Of particular interest are receptorsfor cytokines, molecules that promote the proliferation and/ordifferentiation of cells. Examples of cytokines include erythropoietin(EPO), which stimulates the development of red blood cells;thrombopoietin (TPO), which stimulates development of cells of themegakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia, thrombocytopenia, and neutropenia or receiving chemotherapyfor cancer.

[0004] The demonstrated in vivo activities of these cytokines illustratethe enormous clinical potential of, and need for, other cytokines,cytokine agonists, and cytokine antagonists. The present inventionaddresses these needs by providing new a hematopoletic cytokinereceptor, as well as related compositions and methods.

[0005] The present invention provides such polypeptides for these andother uses that should be apparent to those skilled in the art from theteachings herein.

SUMMARY OF THE INVENTION

[0006] Within one aspect, the present invention provides an isolatedpolynucleotide that encodes a zalphall polypeptide comprising a sequenceof amino acid residues that is at least 90% identical to an amino acidsequence selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 237 (His); (b) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Cys), to amino acid number 255 (Leu);(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 256 (Lys), to amino acid number 538 (Ser); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 538 (Ser); and (e) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 538(Ser), wherein the amino acid percent identity is determined using aFASTA program with ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62, with other parameters setas default. Within one embodiment, the isolated polynucleotide disclosedabove comprises a sequence of polynucleotides that is selected from thegroup consisting of: (a) a polynucleotide sequence as shown in SEQ IDNO:4 from nucleotide 1 to nucleotide 1614; (b) a polynucleotide sequenceas shown in SEQ ID NO:1 from nucleotide 126 to nucleotide 779; (c) apolynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 126 tonucleotide 833; (d) a polynucleotide sequence as shown in SEQ ID NO: 1from nucleotide 834 to nucleotide 1682; (e) a polynucleotide sequence asshown in SEQ ID NO:1 from nucleotide 126 to nucleotide 1682; and (f) apolynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 69 tonucleotide 1682. Within another embodiment, the isolated polynucleotidedisclosed above comprises a sequence of amino acid residues selectedfrom the group consisting of: (a) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 20 (Cys), to amino acid number 237(His); (b) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 20 (Cys), to amino acid number 255 (Leu); (c) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 256 (Lys), toamino acid number 538 (Ser); (d) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Cys), to amino acid number 538 (Ser);and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 1 (Met) to amino acid number 538 (Ser). Within anotherembodiment, the isolated polynucleotide disclosed above consists of asequence of amino acid residues selected from the group consisting of:(a) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 20 (Cys), to amino acid number 237 (His); (b) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 255 (Leu); (c) the amino acid sequence as shown in SEQID NO:2 from amino acid number 256 (Lys), to amino acid number 538(Ser); (d) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 20 (Cys), to amino acid number 538 (Ser); and (e) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) toamino acid number 538 (Ser). Within another embodiment, the isolatedpolynucleotide disclosed above further comprises a WSWSX domain. Withinanother embodiment, the isolated polynucleotide disclosed above furthercomprises a transmembrane domain. Within another embodiment, theisolated polynucleotide disclosed above comprises a transmembrane domainconsisting of residues 238 (Leu) to 255 (Leu) of SEQ ID NO:2. Withinanother embodiment, the isolated polynucleotide disclosed above furthercomprises an intracellular domain. Within another embodiment, theisolated polynucleotide disclosed above comprises an intracellulardomain consists of residues 256 (Lys) to 538 (Ser) of SEQ ID NO:2.Within another embodiment, the isolated polynucleotide disclosed abovecomprises an intracellular domain which domain further comprises Box Iand Box II sites. comprises an intracellular domain wherein thepolypeptide further comprises an affinity tag.

[0007] Within a second aspect, the present invention provides anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a zalphall polypeptidehaving an amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 20 (Cys), to amino acid number 538 (Ser); and a transcriptionterminator, wherein the promoter is operably linked to the DNA segment,and the DNA segment is operably linked to the transcription terminator.

[0008] Within one embodiment, the expression vector disclosed abovefurther comprisies a secretory signal sequence operably linked to theDNA segment.

[0009] Within a third aspect, the present invention provides a culturedcell comprising an expression vector as disclosed above, wherein thecell expresses a polypeptide encoded by the DNA segment.

[0010] Within a fourth aspect, the present invention provides anexpression vector comprising: a transcription promoter; a DNA segmentencoding a zalpha11 polypeptide having an amino acid sequence as shownin SEQ ID NO:2 from amino acid number 20 (Cys), to amino acid number 237(His); and a transcription terminator, wherein the promoter, DNAsegment, and terminator are operably linked. Within one embodiment, theexpression vector disclosed above further comprises a secretory signalsequence operably linked to the DNA segment. Within another embodiment,the expression vector disclosed above further comprises a transmembranedomain operably linked to the DNA segment. Within another embodiment,the expression vector disclosed above further comprises a transmembranedomain consisting of residues 238(Leu) to 255 (Leu) of SEQ ID NO:2.Within another embodiment, the expression vector disclosed above furthercomprises an intracellular domain operably linked to the DNA segment.Within another embodiment, the expression vector disclosed above furthercomprises an intracellular domain consisting of residues 256 (Lys) to538 (Ser) of SEQ ID NO:2.

[0011] Within another aspect, the present invention provides a culturedcell into which has been introduced an expression vector according toclaim 15, wherein the cell expresses a soluble receptor polypeptideencoded by the DNA segment. Within one embodiment, the cultured celldisclosed above is dependent upon an exogenously supplied hematopoieticgrowth factor for proliferation.

[0012] Within another aspect, the present invention provides a DNAconstruct encoding a fusion protein, the DNA construct comprising: afirst DNA segment encoding a polypeptide having a sequence of amino acidresidues selected from the group consisting of: (a) the amino acidsequence of SEQ ID NO:2 from amino acid number 1 (Met), to amino acidnumber 19 (Gly); (b) the amino acid sequence of SEQ ID NO:2 from aminoacid number 20 (Cys) to amino acid number 237 (His); (c) the amino acidsequence of SEQ ID NO:2 from amino acid number 20 (Cys) to amino acidnumber 255 (Leu); (d) the amino acid sequence of SEQ ID NO:2 from aminoacid number 238 (Leu) to amino acid number 255 (Leu); (e) the amino acidsequence of SEQ ID NO:2 from amino acid number 238 (Leu) to amino acidnumber 538 (Ser); (f) the amino acid sequence of SEQ ID NO:2 from aminoacid number 256 (Lys) to amino acid number 538 (Ser); and (g) the aminoacid sequence of SEQ ID NO:2 from amino acid number 20 (Cys), to aminoacid number 538 (Ser); and at least one other DNA segment encoding anadditional polypeptide, wherein the first and other DNA segments areconnected in-frame; and wherein the first and other DNA segments encodethe fusion protein.

[0013] Within another aspect, the present invention provides anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA construct encoding a fusion protein asdisclosed above; and a transcription terminator, wherein the promoter isoperably linked to the DNA construct, and the DNA construct is operablylinked to the transcription terminator.

[0014] Within another aspect, the present invention provides a culturedcell comprising an expression vector as disclosed above, wherein thecell expresses a polypeptide encoded by the DNA construct.

[0015] Within another aspect, the present invention provides a method ofproducing a fusion protein comprising: culturing a cell as disclosedabove; and isolating the polypeptide produced by the cell.

[0016] Within another aspect, the present invention provides an isolatedpolypeptide comprising a sequence of amino acid residues that is atleast 90% identical to an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 237 (His); (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 255 (Leu); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 256 (Lys), to amino acidnumber 538 (Ser); (d) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 538 (Ser); and (e)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met) to amino acid number 538 (Ser), wherein the amino acid percentidentity is determined using a FASTA program with ktup=1, gap openingpenalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62,with other parameters set as default. Within one embodiment, theisolated polypeptide disclosed above comprises a sequence of amino acidresidues selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 237 (His); (b) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Cys), to amino acid number 255 (Leu);(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 256 (Lys), to amino acid number 538 (Ser); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 538 (Ser); and (e) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 538(Ser). Within another embodiment, the isolated polypeptide disclosedabove consists of a sequence of amino acid residues selected from thegroup consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 237 (His); (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 255 (Leu); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 256 (Lys), to amino acidnumber 538 (Ser); (d) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 538 (Ser); and (e)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met) to amino acid number 538 (Ser). Within another embodiment, theisolated polypeptide disclosed above further contains a WSXWS motif.Within another embodiment, the isolated polypeptide disclosed abovefurther comprises a transmembrane domain. Within another embodiment, theisolated polypeptide disclosed above further comprises a transmembranedomain, wherein the transmembrane domain consists of residues 238(Leu)to 255 (Leu) of SEQ ID NO:2. Within another embodiment, the isolatedpolypeptide disclosed above further comprises an intracellular domain.Within another embodiment, the isolated polypeptide disclosed abovefurther comprises an intracellular domain, wherein the intracellulardomain consists of residues 256 (Lys) to 538 (Ser) of SEQ ID NO:2.Within another embodiment, the isolated polypeptide disclosed abovefurther comprises an intracellular domain, wherein the intracellulardomain further comprises Box I and Box II sites.

[0017] Within another aspect, the present invention provides a method ofproducing a zalpha11 polypeptide comprising: culturing a cell asdisclosed above; and isolating the zalpha11 polypeptide produced by thecell.

[0018] Within another aspect, the present invention provides an isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 237 (His); and whereinthe polypeptide is substantially free of transmembrane and intracellulardomains ordinarily associated with hematopoietic receptors. Withinanother embodiment, the isolated polypeptide disclosed above comprisesan affinity tag.

[0019] Within another aspect, the present invention provides a method ofproducing a zalpha11 polypeptide comprising: culturing a cell asdisclosed above; and isolating the zalpha11 polypeptide produced by thecell.

[0020] Within another aspect, the present invention provides a method ofproducing an antibody to zalpha11 polypeptide comprising: inoculating ananimal with a polypeptide selected from the group consisting of: (a) apolypeptide consisting of 9 to 519 amino acids, wherein the polypeptideconsists of a contiguous sequence of amino acids in SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 538 (Ser); (b) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 237 (His); (c) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 101 (Leu) to amino acid number 122 (Gly); (d) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 141 (Asn) to amino acid number 174 (Ala); (e) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 193 (Cys) to amino acid number 261 (Val); (f) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid number 51 (Trp) to amino acid number 61 (Glu); (g) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid 136 (Ile) to amino acid number 143 (Glu); (h) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2 from amino acid 187(Pro) to amino acid number 195 (Ser); (i) a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:2 from amino acid number 223 (Phe)to amino acid number 232 (Glu); and (j) a polypeptide consisting of theamino acid sequence of SEQ ID NO:2 from amino acid number 360 (Glu) toamino acid number 368 (Asp); and wherein the polypeptide elicits animmune response in the animal to produce the antibody; and isolating theantibody from the animal.

[0021] Within another aspect, the present invention provides an antibodyproduced by the method disclosed above, which specifically binds to azalpha11 polypeptide. Within one embodiment, the antibody disclosedabove is a monoclonal antibody.

[0022] Within another aspect, the present invention provides an antibodywhich specifically binds to a polypeptide as disclosed above.

[0023] Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of a modulator of zalpha11protein activity, comprising: culturing a cell into which has beenintroduced an expression vector as disclosed above, wherein the cellexpresses the zalpha11 protein encoded by the DNA segment in thepresence and absence of a test sample; and comparing levels of activityof zalpha11 in the presence and absence of a test sample, by abiological or biochemical assay; and determining from the comparison,the presence of modulator of zalpha11 activity in the test sample.

[0024] Within another aspect, the present invention provides a methodfor detecting a zalpha11 receptor ligand within a test sample,comprising: contacting a test sample with a polypeptide comprising anamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 237 (His); and detecting the binding of thepolypeptide to a ligand in the sample. Within one embodiment, the methoddisclosed above further comprises a polypeptide comprising transmembraneand intracellular domains. Within another embodiment, the methoddisclosed above further comprises a polypeptide wherein the polypeptideis membrane bound within a cultured cell, and the detecting stepcomprises measuring a biological response in the cultured cell. Withinanother embodiment, the method disclosed above further comprises apolypeptide wherein the polypeptide is membrane bound within a culturedcell, and the detecting step comprises measuring a biological responsein the cultured cell, wherein the biological response is cellproliferation or activation of transcription of a reporter gene. Withinanother embodiment, the method disclosed above further comprises apolypeptide wherein the polypeptide is immobilized on a solid support.

[0025] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a Hopp/Woods hydrophilicity plot of human zalpha11.

[0027]FIG. 2 is an alignment of human zalpha11 (zalpha) (SEQ ID NO: 2)and mouse zalpha11 (muzalp) (SEQ ID NO: 85).

DETAILED DESCRIPTION OF THE INVENTION

[0028] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0029] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apolyhistidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0030] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0031] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0032] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0033] The term “complements of a polynucleotide molecule” is apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5° CCCGTGCAT 3′.

[0034] The term “contig” denotes a polynucleotide that has a contiguousstretch of identical or complementary sequence to anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are5′-TAGCTTgagtct-3′ and 3′-gtcgacTACCGA-5′.

[0035] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0036] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0037] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0038] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0039] The term “operably linked”, when referring to DNA segments,indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

[0040] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0041] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, α-globin, β-globin, and myoglobin are paralogs of eachother.

[0042] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

[0043] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0044] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0045] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0046] The term “receptor” is used herein to denote a cell-associatedprotein, or a polypeptide subunit of such a protein, that binds to abioactive molecule (the “ligand”) and mediates the effect of the ligandon the cell. Binding of ligand to receptor results in a conformationalchange in the receptor (and, in some cases, receptor multimerization,i.e., association of identical or different receptor subunits) thatcauses interactions between the effector domain(s) and other molecule(s)in the cell. These interactions in turn lead to alterations in themetabolism of the cell. Metabolic events that are linked toreceptor-ligand interactions include gene transcription,phosphorylation, dephosphorylation, cell proliferation, increases incyclic AMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Cell-surface cytokine receptors arecharacterized by a multi-domain structure as discussed in more detailbelow. These receptors are anchored in the cell membrane by atransmembrane domain characterized by a sequence of hydrophobic aminoacid residues (typically about 21-25 residues), which is commonlyflanked by positively charged residues (Lys or Arg). In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor). The term “receptor polypeptide” is used to denote completereceptor polypeptide chains and portions thereof, including isolatedfunctional domains (e.g., ligand-binding domains).

[0047] A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

[0048] A “soluble receptor” is a receptor polypeptide that is not boundto a cell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

[0049] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0050] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0051] All references cited herein are incorporated by reference intheir entirety.

[0052] The present invention is based in part upon the discovery of anovel DNA sequence that encodes a protein having the structure of aclass I cytokine receptor. The deduced amino acid sequence indicatedthat the encoded receptor belongs to the receptor subfamily thatincludes the IL-2 receptor β-subunit and the β-common receptor (i.e.,IL3, IL-5, and GM-CSF receptor β-subunits). Analysis of the tissuedistribution of the mRNA corresponding to this novel DNA showedexpression in lymph node, peripheral blood leukocytes (PBLs), spleen,and thymus. Moreover, the mRNA was abundant in the Raji cell line (ATCCNo. CCL-86) derived from a Burkitt's lymphoma. The polypeptide has beendesignated zalpha11.

[0053] The novel zalpha11 polypeptides of the present invention wereinitially identified by querying an EST database. An EST was found andits corresponding cDNA was sequenced. The novel polypeptide encoded bythe cDNA showed homology with class I cytokine receptors. The zalpha11polynucleotide sequence encodes the entire coding sequence of thepredicted protein. Zalpha11 is a novel cytokine receptor that may beinvolved in an apoptotic cellular pathway, cell-cell signaling molecule,growth factor receptor, or extracellular matrix associated protein withgrowth factor hormone activity, or the like.

[0054] The sequence of the zalpha11 polypeptide was deduced from asingle clone that contained its corresponding polynucleotide sequence.The clone was obtained from a spinal cord library. Other libraries thatmight also be searched for such sequences include PBL, thymus, spleen,lymph node, human erythroleukemia cell lines (e.g., TF-1), Raji cells,acute monocytic leukemia cell lines, other lymphoid and hematopoieticcell lines, and the like.

[0055] The nucleotide sequence of a representative zalpha11-encoding DNAis described in SEQ ID NO:1 (from nucleotide 69 to 1682), and itsdeduced 538 amino acid sequence is described in SEQ ID NO:2. In itsentirety, the zalpha11 polypeptide (SEQ ID NO:2) represents afull-length polypeptide segment (residue 1 (Met) to residue 538 (Ser) ofSEQ ID NO:2). The domains and structural features of the zalpha11polypeptide are further described below.

[0056] Analysis of the zalpha11 polypeptide encoded by the DNA sequenceof SEQ ID NO:1 revealed an open reading frame encoding 538 amino acids(SEQ ID NO:2) comprising a predicted secretory signal peptide of 19amino acid residues (residue 1 (Met) to residue 19 (Gly) of SEQ IDNO:2), and a mature polypeptide of 519 amino acids (residue 20 (Cys) toresidue 538 (Ser) of SEQ ID NO:2). In addition to the WSXWS motif (SEQID NO:3) corresponding to residues 214 to 218 of SEQ ID NO:2, thereceptor comprises a cytokine-binding domain of approximately 200 aminoacid residues (residues 20 (Cys) to 237 (His) of SEQ ID NO:2); a domainlinker (residues 120 (Pro) to 123 (Pro) of SEQ ID NO:2); a penultimatestrand region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO:2); atransmembrane domain (residues 238 (Leu) to 255 (Leu) of SEQ ID NO:2);complete intracellular signaling domain (residues 256 (Lys) to 538 (Ser)of SEQ ID NO:2) which contains a “Box I” signaling site (residues 267(Ile) to 273 (Pro) of SEQ ID NO:2), and a “Box II” signaling site(residues 301 (Leu) to 304 (Gly) of SEQ ID NO:2). Those skilled in theart will recognize that these domain boundaries are approximate, and arebased on alignments with known proteins and predictions of proteinfolding. In addition to these domains, conserved receptor features inthe encoded receptor include (as shown in SEQ ID NO:2) a conserved Trpresidue at position 138, and a conserved Arg residue at position 201.The corresponding polynucleotides encoding the zalphall polypeptideregions, domains, motifs, residues and sequences described above are asshown in SEQ ID NO: 1.

[0057] The presence of transmembrane regions, and conserved and lowvariance motifs generally correlates with or defines importantstructural regions in proteins. Regions of low variance (e.g.,hydrophobic clusters) are generally present in regions of structuralimportance (Sheppard, P. et al., supra.). Such regions of low varianceoften contain rare or infrequent amino acids, such as Tryptophan. Theregions flanking and between such conserved and low variance motifs maybe more variable, but are often functionally significant because theymay relate to or define important structures and activities such asbinding domains, biological and enzymatic activity, signal transduction,cell-cell interaction, tissue localization domains and the like.

[0058] The regions of conserved amino acid residues in zalphall,described above, can be used as tools to identify new family members.For instance, reverse transcription-polymerase chain reaction (RT-PCR)can be used to amplify sequences encoding the conserved regions from RNAobtained from a variety of tissue sources or cell lines. In particular,highly degenerate primers designed from the zalpha11 sequences areuseful for this purpose. Designing and using such degenerate primers maybe readily performed by one of skill in the art.

[0059] The present invention provides polynucleotide molecules,including DNA and RNA molecules, that encode the zalphall polypeptidesdisclosed herein. Those skilled in the art will recognize that, in viewof the degeneracy of the genetic code, considerable sequence variationis possible among these polynucleotide molecules. SEQ ID NO:4 is adegenerate DNA sequence that encompasses all DNAs that encode thezalpha11 polypeptide of SEQ ID NO:2. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:4 also provides allRNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus,zalpha11 polypeptide-encoding polynucleotides comprising nucleotide 1 tonucleotide 1614 of SEQ ID NO:4 and their RNA equivalents arecontemplated by the present invention. Table 1 sets forth the one-lettercodes used within SEQ ID NO:4 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, Abeing complementary to T, and G being complementary to C. TABLE 1Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T TA A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T WA|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T NA|C|G|T N A|C|G|T

[0060] The degenerate codons used in SEQ ID NO:4, encompassing allpossible codons for a given amino acid, are set forth in Table 2. TABLE2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGT TGYSer S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCACCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN AsnN AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR HisH CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

[0061] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NO:2. Variant sequences can bereadily tested for functionality as described herein.

[0062] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al.Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:4 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

[0063] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences (e.g., >50 base pairs)is performed at temperatures of about 20-25° C. below the calculatedT_(m). For smaller probes (e.g., <50 base pairs) hybridization istypically carried out at the T_(m) or 5-10° C. below. This allows forthe maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.Higher degrees of stringency at lower temperatures can be achieved withthe addition of formamide which reduces the T_(m) of the hybrid about 1°C. for each 1% formamide in the buffer solution. Suitable stringenthybridization conditions are equivalent to about a 5 h to overnightincubation at about 42° C. in a solution comprising: about 40-50%formamide, up to about 6×SSC, about 5×Denhardt's solution, zero up toabout 10% dextran sulfate, and about 10-20 μg/ml denaturedcommercially-available carrier DNA. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide; hybridization is then followed bywashing filters in up to about 2×SSC. For example, a suitable washstringency is equivalent to 0.1×SSC to 2×SSC, 0.1% SDS, at 55° C. to 65°C. Different degrees of stringency can be used during hybridization andwashing to achieve maximum specific binding to the target sequence.Typically, the washes following hybridization are performed atincreasing degrees of stringency to remove non-hybridized polynucleotideprobes from hybridized complexes. Stringent hybridization and washconditions depend on the length of the probe, reflected in the Tm,hybridization and wash solutions used, and are routinely determinedempirically by one of skill in the art.

[0064] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zalpha11 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include PBLs, spleen, thymus, and lymph tissues,Raji cells, human erythroleukemia cell lines (e.g., TF-1), acutemonocytic leukemia cell lines, other lymphoid and hematopoietic celllines, and the like. Total RNA can be prepared using guanidiniumisothiocyanate extraction followed by isolation by centrifugation in aCsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺RNA is prepared from total RNA using the method of Aviv and Leder (Proc.Natl. Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA) isprepared from poly(A)⁺ RNA using known methods. In the alternative,genomic DNA can be isolated. Polynucleotides encoding zalpha11polypeptides are then identified and isolated by, for example,hybridization or polymerase chain reaction (PCR) (Mullis, U.S. Pat. No.4,683,202).

[0065] A full-length clone encoding zalpha11 can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones arepreferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to zalpha11, receptor fragments,or other specific binding partners.

[0066] The polynucleotides of the present invention can also besynthesized using DNA synthesis machines. Currently the method of choiceis the phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short polynucleotides (60 to 80 bp) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. However, for producinglonger polynucleotides (>300 bp), special strategies are usuallyemployed, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length.

[0067] One method for building a synthetic gene requires the initialproduction of a set of overlapping, complementary oligonucleotides, eachof which is between 20 to 60 nucleotides long. Each internal section ofthe gene has complementary 3′ and 5′ terminal extensions designed tobase pair precisely with an adjacent section. Thus, after the gene isassembled, process is completed by sealing the nicks along the backbonesof the two strands with T4 DNA ligase. In addition to the protein codingsequence, synthetic genes can be designed with terminal sequences thatfacilitate insertion into a restriction endonuclease site of a cloningvector. Moreover, other sequences should can be added that containsignals for proper initiation and termination of transcription andtranslation.

[0068] An alternative way to prepare a full-length gene is to synthesizea specified set of overlapping oligonucleotides (40 to 100 nucleotides).After the 3′ and 5′ short overlapping complementary regions (6 to 10nucleotides) are annealed, large gaps still remain, but the shortbase-paired regions are both long enough and stable enough to hold thestructure together. The gaps are filled and the DNA duplex is completedvia enzymatic DNA synthesis by E. coli DNA polymerase I. After theenzymatic synthesis is completed, the nicks are sealed with T4 DNAligase. Double-stranded constructs are sequentially linked to oneanother to form the entire gene sequence which is verified by DNAsequence analysis. See Glick and Pasternak, Molecular Biotechnology,Principles & Applications of Recombinant DNA, (ASM Press, Washington,D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 andClimie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.

[0069] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are zalpha11 polypeptides from other mammalianspecies, including murine, porcine, ovine, bovine, canine, feline,equine, and other primate polypeptides. Orthologs of human zalpha11 canbe cloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses zalpha11 as disclosed herein. Suitable sources ofmRNA can be identified by probing Northern blots with probes designedfrom the sequences disclosed herein. A library is then prepared frommRNA of a positive tissue or cell line. A zalpha11-encoding cDNA canthen be isolated by a variety of methods, such as by probing with acomplete or partial human cDNA or with one or more sets of degenerateprobes based on the disclosed sequences. A cDNA can also be cloned usingPCR (Mullis, supra.), using primers designed from the representativehuman zalpha11 sequence disclosed herein. Within an additional method,the cDNA library can be used to transform or transfect host cells, andexpression of the cDNA of interest can be detected with an antibody tozalpha11 polypeptide. Similar techniques can also be applied to theisolation of genomic clones.

[0070] Cytokine receptor subunits are characterized by a multi-domainstructure comprising an extracellular domain, a transmembrane domainthat anchors the polypeptide in the cell membrane, and an intracellulardomain. The extracellular domain may be a ligand-binding domain, and theintracellular domain may be an effector domain involved in signaltransduction, although ligand-binding and effector functions may resideon separate subunits of a multimeric receptor. The ligand-binding domainmay itself be a multi-domain structure. Multimeric receptors includehomodimers (e.g., PDGF receptor αα and ββ isoforms, erythropoietinreceptor, MPL, and G-CSF receptor), heterodimers whose subunits eachhave ligand-binding and effector domains (e.g., PDGF receptor αβ,isoform), and multimers having component subunits with disparatefunctions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSFreceptors). Some receptor subunits are common to a plurality ofreceptors. For example, the AIC2B subunit, which cannot bind ligand onits own but includes an intracellular signal transduction domain, is acomponent of IL-3 and GM-CSF receptors. Many cytokine receptors can beplaced into one of four related families on the basis of the structureand function. Hematopoietic receptors, for example, are characterized bythe presence of a domain containing conserved cysteine residues and theWSXWS motif (SEQ ID NO:3). Cytokine receptor structure has been reviewedby Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine5:95-106, 1993. Under selective pressure for organisms to acquire newbiological functions, new receptor family members likely arise fromduplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members. Thus, thecytokine receptor superfamily is subdivided into several families, forexample, the immunoglobulin family (including CSF-1, MGF, IL-1, and PDGFreceptors); the hematopoietin family (including IL-2 receptor β-subunit,GM-CSF receptor α-subunit, GM-CSF receptor β-subunit; and G-CSF, EPO,IL-3, IL-4, IL-5, IL-6, IL-7, and IL-9 receptors); TNF receptor family(including TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGFreceptor).

[0071] Analysis of the zalpha11 sequence suggests that it is a member ofthe same receptor subfamily as the IL-2 receptor β-subunit, IL-3, IL-4,and IL-6 receptors. Certain receptors in this subfamily (e.g., G-CSF)associate to form homodimers that transduce a signal. Other members ofthe subfamily (e.g., IL-6, IL-11, and LIF receptors) combine with asecond subunit (termed a β-subunit) to bind ligand and transduce asignal. Specific β-subunits associate with a plurality of specificcytokine receptor subunits. For example, the β-subunit gp130 (Hibi etal., Cell 63:1149-1157, 1990) associates with receptor subunits specificfor IL-6, L-11, and LIF (Gearing et al., EMBO J. 10:2839-2848, 1991;Gearing et al., U.S. Pat. No. 5,284,755). Oncostatin M binds to aheterodimer of LIF receptor and gp130. CNTF binds to trimeric receptorscomprising CNTF receptor, LIF receptor, and gp130 subunits.

[0072] A polynucleotide sequence for the mouse ortholog of humanzalpha11 has been identified and is shown in SEQ ID NO:84 and thecorresponding amino acid sequence shown in SEQ ID NO: 85. Analysis ofthe mouse zalpha11 polypeptide encoded by the DNA sequence of SEQ IDNO:84 revealed an open reading frame encoding 529 amino acids (SEQ IDNO:85) comprising a predicted secretory signal peptide of 19 amino acidresidues (residue 1 (Met) to residue 19 (Ser) of SEQ ID NO:85), and amature polypeptide of 510 amino acids (residue 20 (Cys) to residue 529(Ser) of SEQ ID NO:2). In addition to the WSXWS motif (SEQ ID NO:3)corresponding to residues 214 to 218 of SEQ ID NO:85, the receptorcomprises a cytokine-binding domain of approximately 200 amino acidresidues (residues 20 (Cys) to 237 (His) of SEQ ID NO:85); a domainlinker (residues 120 (Pro) to 123 (Pro) of SEQ ID NO:85); a penultimatestrand region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO:85); atransmembrane domain (residues 238 (Met) to 254 (Leu) of SEQ ID NO:85);complete intracellular signaling domain (residues 255 (Lys) to 529 (Ser)of SEQ ID NO:85) which contains a “Box I” signaling site (residues 266(Ile) to 273 (Pro) of SEQ ID NO:85), and a “Box II” signaling site(residues 301 (Ile) to 304 (Val) of SEQ ID NO:2). A comparison of thehuman and mouse amino acid sequences reveals that both the human andorthologous polypeptides contain corresponding structural featuresdescribed above (See, FIG. 2). The mature sequence for the mousezalpha11 begins at Cys₂₀ (as shown in SEQ ID NO:85), which correspondsto Cys₂₀ (as shown in SEQ ID NO:2) in the human sequence. There is about63% identity between the mouse and human sequences over the entire aminoacid sequence corresponding to SEQ ID NO:2 and SEQ ID NO:85. There isabout 69% identity a between the mouse and human zalpha11 sequences overthe extracellular cytokine binding domain corresponding to residues 20(Cys) to 237 (His) of SEQ ID NO:2 and residues 20 (Cys) to 237 (His) ofSEQ ID NO:85. There is about 60% identity a between the mouse and humanzalpha11 sequences over the intracellular signalling domaincorresponding to residues 256 (Lys) to 538 (Ser) of SEQ ID NO:2, andresidues 255 (Lys) to 529 (Ser) of SEQ ID NO:85. The above percentidentities were determined using a FASTA program with ktup=1, gapopening penalty=12, gap extension penalty=2, and substitutionmatrix=BLOSUM62, with other parameters set as default. The correspondingpolynucleotides encoding the mouse zalpha11 polypeptide regions,domains, motifs, residues and sequences described above are as shown inSEQ ID NO:84.

[0073] Those skilled in the art will recognize that the sequencedisclosed in SEQ ID NO:1 represents a single allele of human zalpha11and that allelic variation and alternative splicing are expected tooccur. Allelic variants of this sequence can be cloned by probing cDNAor genomic libraries from different individuals according to standardprocedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, are within the scope of thepresent invention, as are proteins which are allelic variants of SEQ IDNO:2. cDNAs generated from alternatively spliced mRNAs, which retain theproperties of the zalpha11 polypeptide are included within the scope ofthe present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

[0074] The present invention also provides isolated zalpha11polypeptides that are substantially similar to the polypeptides of SEQID NO:2 and their orthologs. The term “substantially similar” is usedherein to denote polypeptides having at least 70%, more preferably atleast 80%, sequence identity to the sequences shown in SEQ ID NO:2 ortheir orthologs. Such polypeptides will more preferably be at least 90%identical, and most preferably 95% or more identical to SEQ ID NO:2 orits orthologs.) Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919, 1992. Briefly, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 1, and the “blosum 62” scoring matrix of Henikoffand Henikoff (ibid.) as shown in Table 3 (amino acids are indicated bythe standard one-letter codes). The percent identity is then calculatedas:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}}\end{matrix} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0075] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0076] Those skilled in the art appreciate that there are manyestablished algorithms available to align two amino acid sequences. The“FASTA” similarity search algorithm of Pearson and Lipman is a suitableprotein alignment method for examining the level of identity shared byan amino acid sequence disclosed herein and the amino acid sequence of aputative variant zsig57. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

[0077] Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:2) anda test sequence that have either the highest density of identities (ifthe ktup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Preferred parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62, with other parameters setas default. These parameters can be introduced into a FASTA program bymodifying the scoring matrix file (“SMATRIX”), as explained in Appendix2 of Pearson, Meth. Enzymol. 183:63 (1990).

[0078] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from three to six, most preferably three, with otherparameters set as default.

[0079] The BLOSUM62 table (Table 3) is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed below), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3).

[0080] Variant zalpha11 polypeptides or substantially homologouszalpha11 polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-teiminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides of from about 489 to about 568 amino acidresidues that comprise a sequence that is at least 80%, preferably atleast 90%, and more preferably 95% or more identical to thecorresponding region of SEQ ID NO:2. Polypeptides comprising affinitytags can further comprise a proteolytic cleavage site between thezalpha11 polypeptide and the affinity tag. Suitable sites includethrombin cleavage sites and factor Xa cleavage sites. TABLE 4Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0081] The present invention further provides a variety of otherpolypeptide fusions and related multimeric proteins comprising one ormore polypeptide fusions. For example, a zalpha11 polypeptide can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains.Immunoglobulin-zalpha11 polypeptide fusions can be expressed ingenetically engineered cells to produce a variety of multimeric zalpha11analogs. Auxiliary domains can be fused to zalpha11 polypeptides totarget them to specific cells, tissues, or macromolecules (e.g.,collagen). A zalpha11 polypeptide can be fused to two or more moieties,such as an affinity tag for purification and a targeting domain.Polypeptide fusions can also comprise one or more cleavage sites,particularly between domains. See, Tuan et al., Connective TissueResearch 34:1-9, 1996.

[0082] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0083] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for zalpha11 aminoacid residues.

[0084] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, singlealanine mutations are introduced at every residue in the molecule, andthe resultant mutant molecules are tested for biological activity (e.g.ligand binding and signal transduction) as disclosed below to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites ofligand-receptor, protein-protein or other biological interaction canalso be determined by physical analysis of structure, as determined bysuch techniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904,1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities ofessential amino acids can also be inferred from analysis of homologieswith related receptors.

[0085] Determination of amino acid residues that are within regions ordomains that are critical to maintaining structural integrity can bedetermined. Within these regions one can determine specific residuesthat will be more or less tolerant of change and maintain the overalltertiary structure of the molecule. Methods for analyzing sequencestructure include, but are not limited to, alignment of multiplesequences with high amino acid or nucleotide identity and computeranalysis using available software (e.g., the Insight II® viewer andhomology modeling tools; MSI, San Diego, Calif.), secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

[0086] Amino acid sequence changes are made in zalpha11 polypeptides soas to minimize disruption of higher order structure essential tobiological activity. For example, when the zalpha11 polypeptidecomprises one or more helices, changes in amino acid residues will bemade so as not to disrupt the helix geometry and other components of themolecule where changes in conformation abate some critical function, forexample, binding of the molecule to its binding partners. The effects ofamino acid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same disulfide bonding patternas the standard molecule folding would be affected. Another well knownand accepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructural similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

[0087] A Hopp/Woods hydrophilicity profile of the zalpha11 proteinsequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc.Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986and Triquier et al., Protein Engineering 11:153-169, 1998). See, FIG. 1.The profile is based on a sliding six-residue window. Buried G, S, and Tresidues and exposed H, Y, and W residues were ignored. For example, inzalpha11, hydrophilic regions include amino acid residues 55 through 60of SEQ ID NO: 2, amino acid residues 56 through 61 of SEQ ID NO: 2,amino acid residues 139 through 144 of SEQ ID NO: 2, amino acid residues227 through 232 of SEQ ID NO: 2, and amino acid residues 364 through 369of SEQ ID NO: 2.

[0088] Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zalpha11 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include such residues as shown in SEQ ID NO: 2.However, Cysteine residues could be relatively intolerant ofsubstitution.

[0089] The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between class I cytokine receptor familymembers with zalpha11. Using methods such as “FASTA” analysis describedpreviously, regions of high similarity are identified within a family ofproteins and used to analyze amino acid sequence for conserved regions.An alternative approach to identifying a variant zalpha11 polynucleotideon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant zalpha11 polynucleotide canhybridize to a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:1, as discussed above.

[0090] Other methods of identifying essential amino acids in thepolypeptides of the present invention are procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. NatlAcad. Sci. USA 88:4498 (1991), Coombs and Corey, “Site-DirectedMutagenesis and Protein Engineering,” in Proteins: Analysis and Design,Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In thelatter technique, single alanine mutations are introduced at everyresidue in the molecule, and the resultant mutant molecules are testedfor biological activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699 (1996).

[0091] The present invention also includes functional fragments ofzalpha11 polypeptides and nucleic acid molecules encoding suchfunctional fragments. A “functional” zalpha11 or fragment thereofdefined herein is characterized by its proliferative or differentiatingactivity, by its ability to induce or inhibit specialized cellfunctions, or by its ability to bind specifically to an anti-zalpha11antibody or zalpha11 ligand (either soluble or immobilized). Aspreviously described herein, zalpha11 is characterized by a class Icytokine receptor structure. Thus, the present invention furtherprovides fusion proteins encompassing: (a) polypeptide moleculescomprising an extracellular or intracellular domain described herein;and (b) functional fragments comprising one or more of these domains.The other polypeptide portion of the fusion protein may be contributedby another class I cytokine receptor, for exapmple, IL-2 receptorβ-subunit and the β-common receptor (i.e., IL3, IL-5, and GM-CSFreceptor β-subunits), or by a non-native and/or an unrelated secretorysignal peptide that facilitates secretion of the fusion protein.

[0092] Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a zalpha11 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO: 1 or fragments thereof, can bedigested with Bal31 nuclease to obtain a series of nested deletions.These DNA fragments are then inserted into expression vectors in properreading frame, and the expressed polypeptides are isolated and testedfor zalpha11 activity, or for the ability to bind anti-zalpha11antibodies or zalpha11 ligand. One alternative to exonuclease digestionis to use oligonucleotide-directed mutagenesis to introduce deletions orstop codons to specify production of a desired zalpha11 fragment.Alternatively, particular fragments of a zalpha11 polynucleotide can besynthesized using the polymerase chain reaction.

[0093] Standard methods for identifying functional domains arewell-known to those of skill in the art. For example, studies on thetruncation at either or both termini of interferons have been summarizedby Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1 Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

[0094] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/062045) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0095] Variants of the disclosed zalpha11 DNA and polypeptide sequencescan be generated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0096] Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized zalpha11 receptor polypeptides in host cells.Preferred assays in this regard include cell proliferation assays andbiosensor-based ligand-binding assays, which are described below.Mutagenized DNA molecules that encode active receptors or portionsthereof (e.g., ligand-binding fragments, signaling domains, and thelike) can be recovered from the host cells and rapidly sequenced usingmodern equipment. These methods allow the rapid determination of theimportance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

[0097] In addition, the proteins of the present invention (orpolypeptide fragments thereof) can be joined to other bioactivemolecules, particularly other cytokines, to provide multi-functionalmolecules. For example, one or more helices from zalpha11 can be joinedto other cytokines to enhance their biological properties or efficiencyof production.

[0098] The present invention thus provides a series of novel, hybridmolecules in which a segment comprising one or more of the helices ofzalpha11 is fused to another polypeptide. Fusion is preferably done bysplicing at the DNA level to allow expression of chimeric molecules inrecombinant production systems. The resultant molecules are then assayedfor such properties as improved solubility, improved stability,prolonged clearance half-life, improved expression and secretion levels,and pharmacodynamics. Such hybrid molecules may further compriseadditional amino acid residues (e.g. a polypeptide linker) between thecomponent proteins or polypeptides.

[0099] Using the methods discussed herein, one of ordinary skill in theart can identify and/or prepare a variety of polypeptide fragments orvariants of SEQ ID NO:2 that retain the signal transduction or ligandbinding activity. For example, one can make a zalpha11 “solublereceptor” by preparing a variety of polypeptides that are substantiallyhomologous to the cytokine-binding domain (residues 20 (Cys) to 237(His) of SEQ ID NO:2 or allelic variants or species orthologs thereof)and retain ligand-binding activity of the wild-type zalpha11 protein.Such polypeptides may include additional amino acids from, for example,part or all of the transmembrane and intracellular domains. Suchpolypeptides may also include additional polypeptide segments asgenerally disclosed herein such as labels, affinity tags, and the like.

[0100] For any zalpha11 polypeptide, including variants, solublereceptors, and fusion polypeptides or proteins, one of ordinary skill inthe art can readily generate a fully degenerate polynucleotide sequenceencoding that variant using the information set forth in Tables 1 and 2above.

[0101] The zalpha11 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides, can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., New York, 1987.

[0102] In general, a DNA sequence encoding a zalpha11 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0103] To direct a zalpha11 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zalpha11, or may be derivedfrom another secreted protein (e.g., t-PA) or synthesized de novo. Thesecretory signal sequence is operably linked to the zalpha11 DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0104] Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid 1 (Met) toamino acid 19 (Gly) of SEQ ID NO:2 is operably linked to anotherpolypeptide using methods known in the art and disclosed herein. Thesecretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Such constructs have numerous applications known in the art. Forexample, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used in vivo or in vitro to direct peptidesthrough the secretory pathway.

[0105] Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-716, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[0106] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0107] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222 and WIPO publication WO 94/06463. Insect cells can be infectedwith recombinant baculovirus, commonly derived from Autographacalifornica nuclear polyhedrosis virus (AcNPV). See, King, L. A. andPossee, R. D., The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly, D. R. et al., Baculovirus ExpressionVectors: A Laboratory Manual, New York, Oxford University Press., 1994;and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methodsin Molecular Biology, Totowa, N.J., Humana Press, 1995. A second methodof making recombinant zalpha11 baculovirus utilizes a transposon-basedsystem described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,1993). This system, which utilizes transfer vectors, is sold in theBac-to-Bac™ kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, pFastBac1™ (Life Technologies) containing aTn7 transposon to move the DNA encoding the zalpha11 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins, M. S. and Possee, R. D., J Gen Virol71:971-6, 1990; Bonning, B. C. et al., J Gen Virol 75:1551-6, 1994; and,Chazenbalk, G. D., and Rapoport, B., J Biol Chem 270:1543-9, 1995. Inaddition, transfer vectors can include an in-frame fusion with DNAencoding an epitope tag at the C- or N-terminus of the expressedzalpha11 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer,T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a techniqueknown in the art, a transfer vector containing zalphal 1 is transformedinto E. Coli, and screened for bacmids which contain an interrupted lacZgene indicative of recombinant baculovirus. The bacmid DNA containingthe recombinant baculovirus genome is isolated, using common techniques,and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.Recombinant virus that expresses zalpha11 is subsequently produced.Recombinant viral stocks are made by methods commonly used in the art.

[0108] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells.Procedures used are generally described in available laboratory manuals(King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;Richardson, C. D., ibid.). Subsequent purification of the zalpha11polypeptide from the supernatant can be achieved using methods describedherein.

[0109] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondi and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

[0110] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0111] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a zalpha11polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0112] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0113] Within one aspect of the present invention, a zalpha11 cytokinereceptor (including transmembrane and intracellular domains) is producedby a cultured cell, and the cell is used to screen for ligands for thereceptor, including the natural ligand, as well as agonists andantagonists of the natural ligand. To summarize this approach, a cDNA orgene encoding the receptor is combined with other genetic elementsrequired for its expression (e.g., a transcription promoter), and theresulting expression vector is inserted into a host cell. Cells thatexpress the DNA and produce functional receptor are selected and usedwithin a variety of screening systems.

[0114] Mammalian cells suitable for use in expressing the novelreceptors of the present invention and transducing a receptor-mediatedsignal include cells that express a β-subunit, such as gp130, and cellsthat co-express gp130 and LIF receptor (Gearing et al., EMBO J.10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755). In thisregard it is generally preferred to employ a cell that is responsive toother cytokines that bind to receptors in the same subfamily, such asIL-6 or LIF, because such cells will contain the requisite signaltransduction pathway(s). Preferred cells of this type include the humanTF-1 cell line (ATCC number CRL-2003) and the DA-1 cell line (Branch etal., Blood 69:1782, 1987; Broudy et al., Blood 75:1622-1626, 1990). Inthe alternative, suitable host cells can be engineered to produce aβ-subunit or other cellular component needed for the desired cellularresponse. For example, the murine cell line BaF3 (Palacios andSteinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol.6: 4133-4135, 1986), a baby hamster kidney (BHK) cell line, or theCTLL-2 cell line (ATCC TIB-214) can be transfected to express the mousegp130 subunit, or mouse gp130 and LIF receptor, in addition to zalphal1.It is generally preferred to use a host cell and receptor(s) from thesame species, however this approach allows cell lines to be engineeredto express multiple receptor subunits from any species, therebyovercoming potential limitations arising from species specificity. Inthe alternative, species homologs of the human receptor cDNA can becloned and used within cell lines from the same species, such as a mousecDNA in the BaF3 cell line. Cell lines that are dependent upon onehematopoietic growth factor, such as IL-3, can thus be engineered tobecome dependent upon a zalpha11 ligand.

[0115] Cells expressing functional zalpha11 are used within screeningassays. A variety of suitable assays are known in the art. These assaysare based on the detection of a biological response in the target cell.One such assay is a cell proliferation assay. Cells are cultured in thepresence or absence of a test compound, and cell proliferation isdetected by, for example, measuring incorporation of tritiated thymidineor by colorimetric assay based on the metabolic breakdown of AlymarBlue™ (AccuMed, Chicago, Ill.) or3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, 1983). An alternative assay formatuses cells that are further engineered to express a reporter gene. Thereporter gene is linked to a promoter element that is responsive to thereceptor-linked pathway, and the assay detects activation oftranscription of the reporter gene. A preferred promoter element in thisregard is a serum response element, or SRE (see, for example, Shaw etal., Cell 56:563-572, 1989). A preferred such reporter gene is aluciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al., J. Biol. Chem.269:19094-29101, 1994; Schenborn and Goiffin, Promega Notes 41:11,1993). Luciferase assay kits are commercially available from, forexample, Promega Corp., Madison, Wis. Target cell lines of this type canbe used to screen libraries of chemicals, cell-conditioned culturemedia, fungal broths, soil samples, water samples, and the like. Forexample, a bank of cell- or tissue-conditioned media samples can beassayed on a target cell to identify cells that produce ligand. Positivecells are then used to produce a cDNA library in a mammalian cellexpression vector, which is divided into pools, transfected into hostcells, and expressed. Media samples from the transfected cells are thenassayed, with subsequent division of pools, retransfection,subculturing, and re-assay of positive cells to isolate a clonal cellline expressing the ligand. Media samples conditioned by kidney, liver,spleen, thymus, other lymphoid tissues, or T-cells are preferred sourcesof ligand for use in screening procedures.

[0116] A natural ligand for zalpha11 can also be identified bymutagenizing a cytokine-dependent cell line expressing zalpha11 andculturing it under conditions that select for autocrine growth. See WIPOpublication WO 95/21930. Within a typical procedure, cells expressingzalpha11 are mutagenized, such as with EMS. The cells are then allowedto recover in the presence of the required cytokine, then transferred toa culture medium lacking the cytokine. Surviving cells are screened forthe production of a ligand for zalpha11, such as by adding soluble(ligand-binding) receptor polypeptide to the culture medium or byassaying conditioned media on wild-type cells and transfected cellsexpressing the zalpha11. Preferred cell lines for use within this methodinclude cells that are transfected to express gp130 or gp130 incombination with LIF receptor. Preferred such host cell lines includetransfected CTLL-2 cells (Gillis and Smith, Nature 268:154-156, 1977)and transfected BaF3 cells.

[0117] Moreover, a secretion trap method employing zalpha11 solublereceptor polypeptide can be used to isolate a zalpha11 ligand (Aldrich,et al, Cell 87: 1161-1169, 1996). A cDNA expression library preparedfrom a known or suspected ligand source is transfected into COS-7 cells.The cDNA library vector generally has an SV40 origin for amplificationin COS-7 cells, and a CMV promoter for high expression. The transfectedCOS-7 cells are grown in a monolayer and then fixed and permeabilized.Tagged or biotin-labeled zalpha11 soluble receptor, described herein, isthen placed in contact with the cell layer and allowed to bind cells inthe monolayer that express an anti-complementary molecule, i.e., azalpha11 ligand. A cell expressing a ligand will thus be bound withreceptor molecules. An anti-tag antibody (anti-Ig for Ig fusions, M2 oranti-FLAG for FLAG-tagged fusions, streptavidin, and the like) which isconjugated with horseradish peroxidase (HRP) is used to visualize thesecells to which the tagged or biotin-labeled zalpha11 soluble receptorhas bound. The HRP catalyzes deposition of a tyramide reagent, forexample, tyramide-FITC. A commercially-available kit can be used forthis detection (for example, Renaissance TSA-Direct™ Kit; NEN LifeScience Products, Boston, Mass.). Cells which express zalpha11 receptorligand will be identified under fluorescence microscopy as green cellsand picked for subsequent cloning of the ligand using procedures forplasmid rescue as outlined in Aldrich, et al, supra., followed bysubsequent rounds of secretion trap assay until single clones areidentified.

[0118] As a receptor, the activity of zalpha11 polypeptide can bemeasured by a silicon-based biosensor microphysiometer which measuresthe extracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method. See, for example, McConnell, H. M. etal., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,1998; Van Liefde, I. Et al., Eur. J. Pharmacol. 346:87-95, 1998. Themicrophysiometer can be used for assaying eukaryotic, prokaryotic,adherent or non-adherent cells. By measuring extracellular acidificationchanges in cell media over time, the microphysiometer directly measurescellular responses to various stimuli, including agonists, ligands, orantagonists of the zalpha11 polypeptide. Preferably, themicrophysiometer is used to measure responses of a zalpha11-expressingeukaryotic cell, compared to a control eukaryotic cell that does notexpress zalpha11 polypeptide. Zalpha11-expressing eukaryotic cellscomprise cells into which zalpha11 has been transfected, as describedherein, creating a cell that is responsive to zalpha11-modulatingstimuli, or are cells naturally expressing zalpha11, such aszalpha11-expressing cells derived from lymphoid, spleen, thymus tissueor PBLs. Differences, measured by an increase or decrease inextracellular acidification, in the response of cells expressingzalpha11, relative to a control, are a direct measurement ofzalpha11-modulated cellular responses. Moreover, such zalpha11-modulatedresponses can be assayed under a variety of stimuli. Also, using themicrophysiometer, there is provided a method of identifying agonists andantagonists of zalpha11 polypeptide, comprising providing cellsexpressing a zalpha11 polypeptide, culturing a first portion of thecells in the absence of a test compound, culturing a second portion ofthe cells in the presence of a test compound, and detecting an increaseor a decrease in a cellular response of the second portion of the cellsas compared to the first portion of the cells. Antagonists and agonists,including the natural ligand for zalpha11 polypeptide, can be rapidlyidentified using this method.

[0119] Additional assays provided by the present invention include theuse of hybrid receptor polypeptides. These hybrid polypeptides fall intotwo general classes. Within the first class, the intracellular domain ofzalpha11, comprising approximately residues 256 (Lys) to 528 (Ser) ofSEQ ID NO:2, is joined to the ligand-binding domain of a secondreceptor. It is preferred that the second receptor be a hematopoieticcytokine receptor, such as mpl receptor (Souyri et al., Cell63:1137-1147, 1990). The hybrid receptor will further comprise atransmembrane domain, which may be derived from either receptor. A DNAconstruct encoding the hybrid receptor is then inserted into a hostcell. Cells expressing the hybrid receptor are cultured in the presenceof a ligand for the binding domain and assayed for a response. Thissystem provides a means for analyzing signal transduction mediated byzalpha11 while using readily available ligands. This system can also beused to determine if particular cell lines are capable of responding tosignals transduced by zalpha11. A second class of hybrid receptorpolypeptides comprise the extracellular (ligand-binding) domain ofzalpha11 (approximately residues 20 (Cys) to 237 (His) of SEQ ID NO:2)with a cytoplasmic domain of a second receptor, preferably a cytokinereceptor, and a transmembrane domain. The transmembrane domain may bederived from either receptor. Hybrid receptors of this second class areexpressed in cells known to be capable of responding to signalstransduced by the second receptor. Together, these two classes of hybridreceptors enable the use of a broad spectrum of cell types withinreceptor-based assay systems.

[0120] Cells found to express a ligand for zalpha11 are then used toprepare a cDNA library from which the ligand-encoding cDNA may beisolated as disclosed above. The present invention thus provides, inaddition to novel receptor polypeptides, methods for cloning polypeptideligands for the receptors.

[0121] The tissue specificity of zalpha11 expression suggests a role inearly thymocyte development and immune response regulation. Theseprocesses involve stimulation of cell proliferation and differentiationin response to the binding of one or more cytokines to their cognatereceptors. In view of the tissue distribution observed for thisreceptor, agonists (including the natural ligand) and antagonists haveenormous potential in both in vitro and in vivo applications. Compoundsidentified as receptor agonists are useful for stimulating proliferationand development of target cells in vitro and in vivo. For example,agonist compounds are useful as components of defined cell culturemedia, and may be used alone or in combination with other cytokines andhormones to replace serum that is commonly used in cell culture.Agonists are thus useful in specifically promoting the growth and/ordevelopment of T-cells, B-cells, and other cells of the lymphoid andmyeloid lineages, and hematopoietic cells in culture.

[0122] Agonist ligands for zalpha11 may be useful in stimulatingcell-mediated immunity and for stimulating lymphocyte proliferation,such as in the treatment of infections involving immunosuppression,including certain viral infections. Additional uses include tumorsuppression, where malignant transformation results in tumor cells thatare antigenic. Agonist ligands could be used to induce cytotoxicity,which may be mediated through activation of effector cells such asT-cells, NK (natural killer) cells, or LAK (lymphoid activated killer)cells, or induced directly through apoptotic pathways. Agonist ligandsmay also be useful in treating leukopenias by increasing the levels ofthe affected cell type, and for enhancing the regeneration of the T-cellrepertoire after bone marrow transplantation.

[0123] Antagonist ligands or compounds may find utility in thesuppression of the immune system, such as in the treatment of autoimmunediseases, including rheumatoid arthritis, multiple sclerosis, diabetesmellitis, inflammatory bowel disease, Crohn's disease, etc. Immunesuppression can also be used to reduce rejection of tissue or organtransplants and grafts and to treat T-cell specific leukemias orlymphomas by inhibiting proliferation of the affected cell type.

[0124] Zalpha11 may also be used within diagnostic systems for thedetection of circulating levels of ligand. Within a related embodiment,antibodies or other agents that specifically bind to zalpha11 can beused to detect circulating receptor polypeptides. Elevated or depressedlevels of ligand or receptor polypeptides may be indicative ofpathological conditions, including cancer. Soluble receptor polypeptidesmay contribute to pathologic processes and can be an indirect marker ofan underlying disease. For example, elevated levels of soluble IL-2receptor in human serum have been associated with a wide variety ofinflammatory and neoplastic conditions, such as myocardial infarction,asthma, myasthenia gravis, rheumatoid arthritis, acute T-cell leukemia,B-cell lymphomas, chronic lymphocytic leukemia, colon cancer, breastcancer, and ovarian cancer (Heaney et al., Blood 87:847-857, 1996).

[0125] A ligand-binding polypeptide of a zalpha11 receptor, or “solublereceptor,” can be prepared by expressing a truncated DNA encoding thezalpha11 cytokine binding domain (approximately residue 20 (Cys) throughresidue 237 (His) of the human receptor (SEQ ID NO:2)) or thecorresponding region of a non-human receptor. It is preferred that theextracellular domain be prepared in a form substantially free oftransmembrane and intracellular polypeptide segments. Moreover,ligand-binding polypeptide fragments within the zalpha11 cytokinebinding domain, described above, can also serve as zalpha11 solublereceptors for uses described herein. To direct the export of a receptorpolypeptide from the host cell, the receptor DNA is linked to a secondDNA segment encoding a secretory peptide, such as a t-PA secretorypeptide or a zalpha11 secretory peptide. To facilitate purification ofthe secreted receptor polypeptide, a C-terminal extension, such as apoly-histidine tag, substance P, Flag™ peptide (Hopp et al.,Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., NewHaven, Conn.) or another polypeptide or protein for which an antibody orother specific binding agent is available, can be fused to the receptorpolypeptide.

[0126] In an alternative approach, a receptor extracellular domain canbe expressed as a fusion with immunoglobulin heavy chain constantregions, typically an F_(c) fragment, which contains two constant regiondomains and lacks the variable region. Such fusions are typicallysecreted as multimeric molecules wherein the Fc portions are disulfidebonded to each other and two receptor polypeptides are arrayed in closeproximity to each other. Fusions of this type can be used to affinitypurify the cognate ligand from solution, as an in vitro assay tool, toblock signals in vitro by specifically titrating out ligand, and asantagonists in vivo by administering them parenterally to bindcirculating ligand and clear it from the circulation. To purify ligand,a zalpha11-Ig chimera is added to a sample containing the ligand (e.g.,cell-conditioned culture media or tissue extracts) under conditions thatfacilitate receptor-ligand binding (typically near-physiologicaltemperature, pH, and ionic strength). The chimera-ligand complex is thenseparated by the mixture using protein A, which is immobilized on asolid support (e.g., insoluble resin beads). The ligand is then elutedusing conventional chemical techniques, such as with a salt or pHgradient. In the alternative, the chimera itself can be bound to a solidsupport, with binding and elution carried out as above. Collectedfractions can be re-fractionated until the desired level of purity isreached.

[0127] Moreover, zalpha11 soluble receptors can be used as a “ligandsink,” i.e., antagonist, to bind ligand in vivo or in vitro intherapeutic or other applications where the presence of the ligand isnot desired. For example, in cancers that are expressing large amount ofbioactive zalpha11 ligand, zalpha11 soluble receptors can be used as adirect antagonist of the ligand in vivo, and may aid in reducingprogression and symptoms associated with the disease. Moreover, zalpha11soluble receptor can be used to slow the progression of cancers thatover-express zalkpha11 receptors, by binding ligand in vivo that wouldotherwise enhance proliferation of those cancers. Similar in vitroapplications for a zalpha11 soluble receptor can be used, for instance,as a negative selection to select cell lines that grow in the absence ofzalpha11 ligand.

[0128] Moreover, zalpha11 soluble receptor can be used in vivo or indiagnostic applications to detect zalpha11 ligand-expressing cancers invivo or in tissue samples. For example, the zalpha11 soluble receptorcan be conjugated to a radio-label or fluorescent label as describedherein, and used to detect the presence of the ligand in a tissue sampleusing an in vitro ligand-receptor type binding assay, or fluorescentimaging assay. Moreover, a radio-labeled zalpha11 soluble receptor couldbe administered in vivo to detect ligand-expressing solid tumors througha radio-imaging method known in the art.

[0129] Analysis of the tissue distribution of the mRNA corresponding tothis novel DNA showed expression in lymphoid tissues, including thymus,spleen, lymph nodes, and peripheral blood leukocytes. These dataindicate a role for the zalpha11 receptor in proliferation,differentiation, and/or activation of immune cells, and suggest a rolein development and regulation of immune responses. The data also suggestthat the interaction of zalpha11 with its ligand may stimulateproliferation and development of myeloid cells and may, like IL-2, IL-6,LIF, IL-11 and OSM (Baumann et al., J. Biol. Chem. 268:8414-8417, 1993),induce acute-phase protein synthesis in hepatocytes.

[0130] It is preferred to purify the polypeptides of the presentinvention to ≧80% purity, more preferably to >90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

[0131] Expressed recombinant zalpha11 polypeptides (or zalpha11 chimericor fusion polypeptides) can be purified using fractionation and/orconventional purification methods and media. Ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

[0132] The polypeptides of the present invention can be isolated byexploitation of their biochemical, structural, and biologicalproperties. For example, immobilized metal ion adsorption (IMAC)chromatography can be used to purify histidine-rich proteins, includingthose comprising polyhistidine tags. Briefly, a gel is first chargedwith divalent metal ions to form a chelate (Sulkowski, Trends inBiochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to thismatrix with differing affinities, depending upon the metal ion used, andwill be eluted by competitive elution, lowering the pH, or use of strongchelating agents. Other methods of purification include purification ofglycosylated proteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), Acad. Press, San Diego, 1990,pp.529-39). Within additional embodiments of the invention, a fusion ofthe polypeptide of interest and an affinity tag (e.g., maltose-bindingprotein, an immunoglobulin domain) may be constructed to facilitatepurification.

[0133] Moreover, using methods described in the art, polypeptidefusions, or hybrid zalpha11 proteins, are constructed using regions ordomains of the inventive zalpha11 in combination with those of otherhuman cytokine receptor family proteins, or heterologous proteins(Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin.Biology, 5:511-5, 1994, and references therein). These methods allow thedetermination of the biological importance of larger domains or regionsin a polypeptide of interest. Such hybrids may alter reaction kinetics,binding, constrict or expand the substrate specificity, or alter tissueand cellular localization of a polypeptide, and can be applied topolypeptides of unknown structure.

[0134] Fusion polypeptides or proteins can be prepared by methods knownto those skilled in the art by preparing each component of the fusionprotein and chemically conjugating them. Alternatively, a polynucleotideencoding one or more components of the fusion protein in the properreading frame can be generated using known techniques and expressed bythe methods described herein. For example, part or all of a domain(s)conferring a biological function may be swapped between zalpha11 of thepresent invention with the functionally equivalent domain(s) fromanother cytokine family member. Such domains include, but are notlimited to, the secretory signal sequence, extracellular cytokinebinding domain, transmembrane domain, and intracellular signalingdomain, Box I and Box II sites, as disclosed herein. Such fusionproteins would be expected to have a biological functional profile thatis the same or similar to polypeptides of the present invention or otherknown family proteins, depending on the fusion constructed. Moreover,such fusion proteins may exhibit other properties as disclosed herein.

[0135] Standard molecular biological and cloning techniques can be usedto swap the equivalent domains between the zalpha11 polypeptide andthose polypeptides to which they are fused. Generally, a DNA segmentthat encodes a domain of interest, e.g., a zalpha11 domain describedherein, is operably linked in frame to at least one other DNA segmentencoding an additional polypeptide (for instance a domain or region fromanother cytokine receptor, such as the IL-2 receptor), and inserted intoan appropriate expression vector, as described herein. Generally DNAconstructs are made such that the several DNA segments that encode thecorresponding regions of a polypeptide are operably linked in frame tomake a single construct that encodes the entire fusion protein, or afunctional portion thereof. For example, a DNA construct would encodefrom N-terminus to C-terminus a fusion protein comprising a signalpolypeptide followed by a cytokine binding domain, followed by atransmembrane domain, followed by an intracellular signaling domain.Such fusion proteins can be expressed, isolated, and assayed foractivity as described herein.

[0136] Zalpha11 polypeptides or fragments thereof may also be preparedthrough chemical synthesis. zalpha11 polypeptides may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;and may or may not include an initial methionine amino acid residue.

[0137] Polypeptides of the present invention can also be synthesized byexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. Methods for synthesizingpolypeptides are well known in the art. See, for example, Merrifield, J.Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595,1970. After the entire synthesis of the desired peptide on a solidsupport, the peptide-resin is with a reagent which cleaves thepolypeptide from the resin and removes most of the side-chain protectinggroups. Such methods are well established in the art.

[0138] The activity of molecules of the present invention can bemeasured using a variety of assays that measure cell differentiation andproliferation. Such assays are well known in the art.

[0139] Proteins of the present invention are useful for example, intreating lymphoid, immune, inflammatory, spleenic, blood or bonedisorders, and can be measured in vitro using cultured cells or in vivoby administering molecules of the claimed invention to the appropriateanimal model. For instance, host cells expressing a zalpha11 solublereceptor polypeptide can be embedded in an alginate environment andinjected (implanted) into recipient animals. Alginate-poly-L-lysinemicroencapsulation, permselective membrane encapsulation and diffusionchambers are a means to entrap transfected mammalian cells or primarymammalian cells. These types of non-immunogenic “encapsulations” permitthe diffusion of proteins and other macromolecules secreted or releasedby the captured cells to the recipient animal. Most importantly, thecapsules mask and shield the foreign, embedded cells from the recipientanimal's immune response. Such encapsulations can extend the life of theinjected cells from a few hours or days (naked cells) to several weeks(embedded cells). Alginate threads provide a simple and quick means forgenerating embedded cells.

[0140] The materials needed to generate the alginate threads are knownin the art. In an exemplary procedure, 3% alginate is prepared insterile H₂O, and sterile filtered. Just prior to preparation of alginatethreads, the alginate solution is again filtered. An approximately 50%cell suspension (containing about 5×10⁵ to about 5×10⁷ cells/ml) ismixed with the 3% alginate solution. One ml of the alginate/cellsuspension is extruded into a 100 mM sterile filtered CaCl₂ solutionover a time period of ˜15 min, forming a “thread”. The extruded threadis then transferred into a solution of 50 mM CaCl₂, and then into asolution of 25 mM CaCl₂. The thread is then rinsed with deionized waterbefore coating the thread by incubating in a 0.01% solution ofpoly-L-lysine. Finally, the thread is rinsed with Lactated Ringer'sSolution and drawn from solution into a syringe barrel (without needle).A large bore needle is then attached to the syringe, and the thread isintraperitoneally injected into a recipient in a minimal volume of theLactated Ringer's Solution.

[0141] An in vivo approach for assaying proteins of the presentinvention involves viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, retroviruses, vaccinia virus,and adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: (i) adenovirus can accommodate relatively large DNA inserts;(ii) can be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) can be used with a large number of differentpromoters including ubiquitous, tissue specific, and regulatablepromoters. Also, because adenoviruses are stable in the bloodstream,they can be administered by intravenous injection.

[0142] Using adenovirus vectors where portions of the adenovirus genomeare deleted, inserts are incorporated into the viral DNA by directligation or by homologous recombination with a co-transfected plasmid.In an exemplary system, the essential E1 gene has been deleted from theviral vector, and the virus will not replicate unless the E1 gene isprovided by the host cell (the human 293 cell line is exemplary). Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an El genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined.

[0143] Moreover, adenoviral vectors containing various deletions ofviral genes can be used in an attempt to reduce or eliminate immuneresponses to the vector. Such adenoviruses are E1 deleted, and inaddition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol.72:2022-2032, 1998; Raper, S. E. et al., Human Gene Therapy 9:671-679,1998). In addition, deletion of E2b is reported to reduce immuneresponses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998). Moreover,by deleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses where all viral genes are deleted are particularlyadvantageous for insertion of large inserts of heterologous DNA. Forreview, see Yeh, P. and Perricaudet, M., FASEB J. 11:615-623, 1997.

[0144] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293 cells can be grown as adherent cells orin suspension culture at relatively high cell density to producesignificant amounts of protein (See Garnier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins may also be effectivelyobtained.

[0145] In view of the tissue distribution observed for zalpha11,agonists (including the natural ligand/substrate/cofactor/etc.) andantagonists have enormous potential in both in vitro and in vivoapplications. Compounds identified as zalpha11 agonists are useful forstimulating growth of immune and hematopoietic cells in vitro and invivo. For example, zalpha11 and agonist compounds are useful ascomponents of defined cell culture media, and may be used alone or incombination with other cytokines and hormones to replace serum that iscommonly used in cell culture. Agonists are thus useful in specificallypromoting the growth and/or development of T-cells, B-cells, and othercells of the lymphoid and myeloid lineages in culture. Moreover,zalpha11 soluble receptor, agonist, or antagonist may be used in vitroin an assay to measure stimulation of colony formation from isolatedprimary bone marrow cultures. Such assays are well known in the art.

[0146] Antagonists are also useful as research reagents forcharacterizing sites of ligand-receptor interaction. Inhibitors ofzalpha11 activity (zalpha11 antagonists) include anti-zalpha11antibodies and soluble zalpha11 receptors, as well as other peptidic andnon-peptidic agents (including ribozymes).

[0147] Zalpha11 can also be used to identify modulators (e.g,antagonists) of its activity. Test compounds are added to the assaysdisclosed herein to identify compounds that inhibit the activity ofzalpha11. In addition to those assays disclosed herein, samples can betested for inhibition of zalpha11 activity within a variety of assaysdesigned to measure zalpha11 binding, oligomerization, or thestimulation/inhibition of zalpha11-dependent cellular responses. Forexample, zalpha11-expressing cell lines can be transfected with areporter gene construct that is responsive to a zalpha11-stimulatedcellular pathway. Reporter gene constructs of this type are known in theart, and will generally comprise a zalpha11-DNA response elementoperably linked to a gene encoding an assay detectable protein, such asluciferase. DNA response elements can include, but are not limited to,cyclic AMP response elements (CRE), hormone response elements (HRE)insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Scd.USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestleret al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec.Endocrinol. 4 (8):1087-94; 1990. Hormone response elements are reviewedin Beato, Cell 56:335-44; 1989. Candidate compounds, solutions, mixturesor extracts or conditioned media from various cell types are tested forthe ability to enhance the activity of zalpha11 receptor as evidenced bya increase in zalpha11 stimulation of reporter gene expression. Assaysof this type will detect compounds that directly stimulate zalpha11signal transduction activity through binding the receptor or byotherwise stimulating part of the signal cascade. As such, there isprovided a method of identifying agonists of zalpha11 polypeptide,comprising providing cells responsive to a zalpha11 polypeptide,culturing a first portion of the cells in the absence of a testcompound, culturing a second portion of the cells in the presence of atest compound, and detecting a increase in a cellular response of thesecond portion of the cells as compared to the first portion of thecells. Moreover third cell, containing the reporter gene constructdescribed above, but not expressing zaplpha11 receptor, can be used as acontrol cell to assess non-specific, or ndn-zalpiha11-mediated,stimulation of the reporter. Agonists, including the natural ligand, aretherefore useful to stimulate or increase zalpha11 polypeptide function.

[0148] A zalpha11 ligand-binding polypeptide, such as the cytokinebinding domain disclosed herein, can also be used for purification ofligand. The polypeptide is immobilized on a solid support, such as beadsof agarose, cross-linked agarose, glass, cellulosic resins, silica-basedresins, polystyrene, cross-linked polyacrylamide, or like materials thatare stable under the conditions of use. Methods for linking polypeptidesto solid supports are known in the art, and include amine chemistry,cyanogen bromide activation, N-hydroxysuccinimide activation, epoxideactivation, sulfhydryl activation, and hydrazide activation. Theresulting medium will generally be configured in the form of a column,and fluids containing ligand are passed through the column one or moretimes to allow ligand to bind to the receptor polypeptide. The ligand isthen eluted using changes in salt concentration, chaotropic agents(guanidine HCl), or pH to disrupt ligand-receptor binding.

[0149] An assay system that uses a ligand-binding receptor (or anantibody, one member of a complement/ anti-complement pair) or a bindingfragment thereof, and a commercially available biosensor instrument maybe advantageously employed (e.g., BIAcore™, Pharmacia Biosensor,Piscataway, N.J.; or SELDI™ technology, Ciphergen, Inc., Palo Alto,Calif.). Such receptor, antibody, member of a complement/anti-complementpair or fragment is immobilized onto the surface of a receptor chip. Useof this instrument is disclosed by Karlsson, J. Immunol. Methods145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63,1993. A receptor, antibody, member or fragment is covalently attached,using amine or sulfhydryl chemistry, to dextran fibers that are attachedto gold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

[0150] Ligand-binding receptor polypeptides can also be used withinother assay systems known in the art. Such systems include Scatchardanalysis for determination of binding affinity (see Scatchard, Ann. NYAcad. Sci. 51: 660-672, 1949) and calorimetric assays (Cunningham etal., Science 253:545-48, 1991; Cunningham et al., Science 245:821-25,1991).

[0151] Zalpha11 polypeptides can also be used to prepare antibodies thatbind to zalpha11 epitopes, peptides or polypeptides. The zalpha11polypeptide or a fragment thereof serves as an antigen (immunogen) toinoculate an animal and elicit an immune response. One of skill in theart would recognize that antigens or immunogenic epitopes can consist ofstretches of amino acids within a longer polypeptide, from about 10amino acids and up to about the entire length of the polypeptide orlonger depending on the polypeptide. Suitable antigens include thezalpha11 polypeptide encoded by SEQ ID NO:2 from amino acid number 20(Cys) to amino acid number 538 (Ser), or a contiguous 9 to 519 AA aminoacid fragment thereof. Preferred peptides to use as antigens are thecytokine binding domain, intracellular signaling domain, Box I and BoxII sites, disclosed herein, and zalpha11 hydrophilic peptides such asthose predicted by one of skill in the art from a hydrophobicity plot,determined for example, from a Hopp/Woods hydrophilicity profile basedon a sliding six-residue window, with buried G, S, and T residues andexposed H, Y, and W residues ignored (See, FIG. 1). Zalpha11 hydrophilicpeptides include peptides comprising amino acid sequences selected fromthe group consisting of: (1) amino acid number 51 (Trp) to amino acidnumber 61 (Glu) of SEQ ID NO:2; (2) amino acid number 136 (Ile) to aminoacid number 143 (Glu) of SEQ ID NO:2; (3) amino acid number 187 (Pro) toamino acid number 195 (Ser) of SEQ ID NO:2; (4) amino acid number 223(Phe) to amino acid number 232 (Glu) of SEQ ID NO:2; and (5) amino acidnumber 360 (Glu) to amino acid number 368 (Asp) of SEQ ID NO:2. Inaddition, conserved motifs, and variable regions between conservedmotifs of zalpha11 are suitable antigens. Moreover, correspondingregions of the mouse zalpha11 polypeptide (SEQ ID NO:85) can be used togenerate antibodies against the mouse zalpha11. Antibodies generatedfrom this immune response can be isolated and purified as describedherein. Methods for preparing and isolating polyclonal and monoclonalantibodies are well known in the art. See, for example, CurrentProtocols in Immunology, Cooligan, et al. (eds.), National Institutes ofHealth, John Wiley and Sons, Inc., 1995; Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982.

[0152] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, and rats with a zalpha11 polypeptide or a fragmentthereof. The immunogenicity of a zalpha11 polypeptide may be increasedthrough the use of an adjuvant, such as alum (aluminum hydroxide) orFreund's complete or incomplete adjuvant. Polypeptides useful forimmunization also include fusion polypeptides, such as fusions ofzalpha11 or a portion thereof with an immunoglobulin polypeptide or withmaltose binding protein. The polypeptide immunogen may be a full-lengthmolecule or a portion thereof. If the polypeptide portion is“hapten-like”, such portion may be advantageously joined or linked to amacromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA) or tetanus toxoid) for immunization.

[0153] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human. framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

[0154] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to zalpha11protein or peptide, and selection of antibody display libraries in phageor similar vectors (for instance, through use of immobilized or labeledzalpha11 protein or peptide). Genes encoding polypeptides havingpotential zalpha11 polypeptide binding domains can be obtained byscreening random peptide libraries displayed on phage (phage display) oron bacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al.,U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using thezalpha11 sequences disclosed herein to identify proteins which bind tozalpha11. These “binding peptides” which interact with zalpha11polypeptides can be used for tagging cells; for isolating homologpolypeptides by affinity purification; they can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like.These binding peptides can also be used in analytical methods such asfor screening expression libraries and neutralizing activity. Thebinding peptides can also be used for diagnostic assays for determiningcirculating levels of zalpha11 polypeptides; for detecting orquantitating soluble zalpha11 polypeptides as marker of underlyingpathology or disease. These binding peptides can also act as zalpha11“antagonists” to block zalpha11 binding and signal transduction in vitroand in vivo. These anti-zalpha11 binding peptides would be useful forinhibiting the action of a ligand that binds with zalpha11.

[0155] Antibodies are determined to be specifically binding if: 1) theyexhibit a threshold level of binding activity, and/or 2) they do notsignificantly cross-react with related polypeptide molecules. First,antibodies herein specifically bind if they bind if they bind to azalpha11 polypeptide, peptide or epitope with an affinity at least10-fold greater than the binding affinity to control (non-zalpha11)polypeptide. It is preferred that the antibodies exhibit a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).

[0156] Second, antibodies are determined to specifically bind if they donot significantly cross-react with related polypeptides. Antibodies donot significantly cross-react with related polypeptide molecules, forexample, if they detect zalpha11 but not known related polypeptidesusing a standard Western blot analysis (Ausubel et al., ibid.). Examplesof known related polypeptides are orthologs, proteins from the samespecies that are members of a protein family (e.g. IL-6), zalpha11polypeptides, and non-human zalpha11. Moreover, antibodies may be“screened against” known related polypeptides to isolate a populationthat specifically binds to the inventive polypeptides. For example,antibodies raised to zalpha11 are adsorbed to related polypeptidesadhered to insoluble matrix; antibodies specific to zalpha11 will flowthrough the matrix under the proper buffer conditions. Such screeningallows isolation of polyclonal and monoclonal antibodiesnon-crossreactive to closely related polypeptides (Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995).Screening and isolation of specific antibodies is well known in the art.See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff etal., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principlesand Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjaminet al., Ann. Rev. Immunol. 2: 67-101, 1984.

[0157] A variety of assays known to those skilled in the art can beutilized to detect antibodies which specifically bind to zalpha11proteins or peptides. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1988. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant zalpha11 protein or polypeptide.

[0158] Antibodies to zalpha11 may be used for tagging cells that expresszalpha11; for isolating zalpha11 by affinity purification; fordiagnostic assays for determining circulating levels of zalpha11polypeptides; for detecting or quantitating soluble zalpha11 as markerof underlying pathology or disease; in analytical methods employingFACS; for screening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzalpha11 activity in vitro and in vivo. Suitable direct tags or labelsinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. Antibodiesherein may also be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to zalpha11or fragments thereof may be used in vitro to detect denatured zalpha11or fragments thereof in assays, for example, Western Blots or otherassays known in the art.

[0159] Antibodies to zalpha11 are useful for tagging cells that expressthe receptor and assaying Zalpha11 expression levels, for affinitypurification, within diagnostic assays for determnining circulatinglevels of soluble receptor polypeptides, analytical methods employingfluorescence-activated cell sorting. Divalent antibodies may be used asagonists to mimic the effect of the zalpha11 ligand.

[0160] Antibodies herein can also be directly or indirectly conjugatedto drugs, toxins, radionuclides and the like, and these conjugates usedfor in vivo diagnostic or therapeutic applications. For instance,antibodies or binding polypeptides which recognize zalpha11 of thepresent invention can be used to identify or treat tissues or organsthat express a corresponding anti-complementary molecule (i.e., azalpha11 receptor). More specifically, anti-zalpha11 antibodies, orbioactive fragments or portions thereof, can be coupled to detectable orcytotoxic molecules and delivered to a mammal having ceils, tissues ororgans that express the zalpha11 molecule.

[0161] Suitable detectable molecules may be directly or indirectlyattached to polypeptides that bind zalpha11 (“binding polypeptides,”including binding peptides disclosed above), antibodies, or bioactivefragments or portions thereof. Suitable detectable molecules includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmarkers, chemiluminescent markers, magnetic particles and the like.Suitable cytotoxic molecules may be directly or indirectly attached tothe polypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Binding polypeptides or antibodies may also be conjugatedto cytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the binding polypeptide orantibody portion. For these purposes, biotin/streptavidin is anexemplary complementary/anticomplementary pair.

[0162] In another embodiment, binding polypeptide-toxin fusion proteinsor antibody-toxin fusion proteins can be used for targeted cell ortissue inhibition or ablation (for instance, to treat cancer cells ortissues). Alternatively, if the binding polypeptide has multiplefunctional domains (i.e., an activation domain or a ligand bindingdomain, plus a targeting domain), a fusion protein including only thetargeting domain may be suitable for directing a detectable molecule, acytotoxic molecule or a complementary molecule to a cell or tissue typeof interest. In instances where the fusion protein including only asingle domain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

[0163] In another embodiment, zalpha11 binding. polypeptide-cytokine orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, blood, lymphoid, colon, and bonemarrow cancers), if the binding polypeptide-cytokine or anti-zalpha11antibody targets the hyperproliferative cell (See, generally, Hornick etal., Blood 89:4437-47, 1997). They described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of, cytokine. Suitable anti-zalpha11antibodies target an undesirable cell or tissue (i.e., a tumor or aleukemia), and the fused cytokine mediates improved target cell lysis byeffector cells. Suitable cytokines for this purpose include interleukin2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), forinstance.

[0164] Alternatively, zalpha11 binding polypeptide or antibody fusionproteins described herein can be used for enhancing in vivo killing oftarget tissues by directly stimulating a zalpha11-modulated apoptoticpathway, resulting in cell death of hyperproliferative cells expressingzalpha11.

[0165] The bioactive binding polypeptide or antibody conjugatesdescribed herein can be delivered orally, intravenously, intraarteriallyor intraductally, or may be introduced locally at the intended site ofaction.

[0166] Four-helix bundle cytokines that bind to cytokine receptors aswell as other proteins produced by activated lymphocytes play animportant biological role in cell differentiation, activation,recruitment and homeostasis of cells throughout the body. Therapeuticutility includes treatment of diseases which require immune regulationincluding autoimmune diseases, such as, rheumatoid arthritis, multiplesclerosis, myasthenia gravis, systemic lupus erythomatosis and diabetes.Zalpha11 antagonists or agonists, including soluble receptors and thenatural ligand, may be important in the regulation of inflammation, andtherefore would be useful in treating rheumatoid arthritis, asthma,ulcerative colitis, inflammatory bowel disease, Crohn's disease, andsepsis. There may be a role of zalpha11 antagonists or agonists,including soluble receptors and the natural ligand, in mediatingtumorgenesis, and therefore would be useful in the treatment of cancer.Zalpha11 antagonists or agonists, including soluble receptors and thenatural ligand, may be a potential therapeutic in suppressing the immunesystem which would be important for reducing graft rejection. Zalpha11Ligand may have usefulness in prevention of graft vs. host disease.

[0167] Alternatively, zalpha11 antagonists or agonists, includingsoluble receptors and the natural ligand may activate the immune systemwhich would be important in boosting immunity to infectious diseases,treating immunocompromised patients, such as HIV+ patient, or inimproving vaccines. In particular, zalpha11 antagonists or agonists,including soluble receptors and the natural ligand can modulate,stimulate or expand NK cells, or their progenitors, and would providetherapeutic value in treatment of viral infection, and as ananti-neoplastic factor. NK cells are thought to play a major role inelimination of metastatic tumor cells and patients with both metastasesand solid tumors have decreased levels of NK cell activity (Whitesideet. al., Curr. Top. Microbiol. Immunol. 230:221-244, 1998).

[0168] Polynucleotides encoding zalpha11 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzalpha11 activity. If a mammal has a mutated or absent zalpha11 gene,the zalpha11 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zalpha11 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0169] In another embodiment, a zalpha11 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

[0170] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

[0171] Antisense methodology can be used to inhibit zalpha11 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of azalpha11-encoding polynucleotide (e.g., a polynucleotide as set froth inSEQ ID NO: 1) are designed to bind to zalpha11-encoding mRNA and toinhibit translation of such mRNA. Such antisense polynucleotides areused to inhibit expression of zalpha11 polypeptide-encoding genes incell culture or in a subject.

[0172] In addition, as a cell surface molecule, zalpha11 polypeptide canbe used as a target to introduce gene therapy into a cell. Thisapplication would be particularly appropriate for introducingtherapeutic genes into cells in which zalpha11 is normally expressed,such as lymphoid tissue and PBLs, or cancer cells which express zalpha11polypeptide. For example, viral gene therapy, such as described above,can be targeted to specific cell types in which express a cellularreceptor, such as zalpha11 polypeptide, rather than the viral receptor.Antibodies, or other molecules that recognize zalpha11 molecules on thetarget cell's surface can be used to direct the virus to infect andadminister gene therapeutic material to that target cell. See, Woo, S.L. C, Nature Biotech. 14:1538, 1996; Wickham, T. J. et al, NatureBiotech. 14:1570-1573, 1996; Douglas, J. T et al., Nature Biotech.14:1574-1578, 1996; Rihova, B., Crit. Rev. Biotechnol. 17:149-169, 1997;and Vile, R. G. et al., Mol. Med. Today 4:84-92, 1998. For example, abispecific antibody containing a virus-neutralizing Fab fragment coupledto a zalpha11-specific antibody can be used to direct the virus to cellsexpressing the zalpha11 receptor and allow efficient entry of the viruscontaining a genetic element into the cells. See, for example, Wickham,T. J., et al., J. Virol. 71:7663-7669, 1997; and Wickham, T. J., et al.,J. Virol. 70:6831-6838, 1996.

[0173] The present invention also provides reagents which will find usein diagnostic applications. For example, the zalpha11 gene, a probecomprising zalpha11 DNA or RNA or a subsequence thereof can be used todetermine if the zalpha11 gene is present on chromosome 16 or if amutation has occurred. Zalpha11 is located at the 16p11.1 region ofchromosome 16 (See, Example 3). Detectable chromosomal aberrations atthe zalpha11 gene locus include, but are not limited to, aneuploidy,gene copy number changes, insertions, deletions, restriction sitechanges and rearrangements. Such aberrations can be detected usingpolynucleotides of the present invention by employing molecular genetictechniques, such as restriction fragment length polymorphism (RFLP)analysis, fluorescence in situ hybridization methods, short tandemrepeat (STR) analysis employing PCR techniques, and other geneticlinkage analysis techniques known in the art (Sambrook et al., ibid.;Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).

[0174] The precise knowledge of a gene's position can be useful for anumber of purposes, including: 1) determining if a sequence is part ofan existing contig and obtaining additional surrounding geneticsequences in various forms, such as YACs, BACs or cDNA clones; 2)providing a possible candidate gene for an inheritable disease whichshows linkage to the same chromosomal region; and 3) cross-referencingmodel organisms, such as mouse, which may aid in determining whatfunction a particular gene might have.

[0175] The zalpha11 gene is located at the 16p11.1 region of chromosome16. Several genes of known function map to this region. For example, theinterleukin 4 (IL-4) cytokine receptor alpha-subunit, a member of thehematopoietin receptor family, maps to 16p12.1-p11.2. This subunit mayform a heterodimer with zalpha11. Moreover, zalpha11 polynucleotideprobes can be used to detect abnormalities or genotypes associated withdefects in IL-4 receptor, such as those that are implicated in someallergic inflammatory disorders and asthma (Deichman, K. A. et al., Exp.Allergy 28:151-155; 1998; Mitsuyasu, H. et al., Nature Genet.19:119-120, 1998). In addition, zalpha11 polynucleotide probes can beused to detect abnormalities or genotypes associated with inflammatorybowel disease, where a susceptibility marker maps to 16p12-q13 (Cho, J.H. et al, Proc. Nat. Acad. Sci. 95:7502-7507, 1998). Further, zalpha11polynucleotide probes can be used to detect abnormalities or genotypesassociated with hemoglobin loci located at 16pter-p13.3; andparticularly hemoglobin-alpha defects associated with alpha-thalassemiasyndromes, such as hydrops fetalis (for review, see Chui, M. P., andWaye, J. S. Blood 91:2213-2222, 1998). Moreover, amongst other geneticloci, those for Wilms tumor, type III (16q), Rubenstein-Taybi syndrome(16p13.3), severe infantile polycystic kidney disease (16p13.3), allmanifest themselves in human disease states as well as map to thisregion of the human genome. See the Online Mendellian Inheritance of Man(OMIM) gene map, and references therein, for this region of chromosome16 on a publicly available WWW server(http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=16p11.1).All of these serve as possible candidate genes for an inheritabledisease which show linkage to the same chromosomal region as thezalpha11 gene.

[0176] Similarly, defects in the zalpha11 locus itself may result in aheritable human disease state. Molecules of the present invention, suchas the polypeptides, antagonists, agonists, polynucleotides andantibodies of the present invention would aid in the detection,diagnosis prevention, and treatment associated with a zalpha11 geneticdefect.

[0177] Mice engineered to express the zalpha11 gene, referred to as“transgenic mice,” and mice that exhibit a complete absence of zalpha11gene function, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express zalpha11, either ubiquitously or undera tissue-specific or tissue-restricted promoter can be used to askwhether over-expression causes a phenotype. For example, over-expressionof a wild-type zalpha11 polypeptide, polypeptide fragment or a mutantthereof may alter normal cellular processes, resulting in a phenotypethat identifies a tissue in which zalpha11 expression is functionallyrelevant and may indicate a therapeutic target for the zalpha11, itsagonists or antagonists. For example, a preferred transgenic mouse toengineer is one that expresses a “dominant-negative” phenotype, such asone that over-expresses the zalpha11 extracellular cytokine bindingdomain with the transmembrane domain attached (approximately amino acids20 (Cys) to 255 (Leu) of SEQ ID NO:2). Moreover, such over-expressionmay result in a phenotype that shows similarity with human diseases.Similarly, knockout zalpha11 mice can be used to determine wherezalpha11 is absolutely required in vivo. The phenotype of knockout miceis predictive of the in vivo effects of that a zalpha11 antagonist, suchas those described herein, may have. The mouse or the human zalpha11cDNA can be used to isolate murine zalpha11 mRNA, cDNA and genomic DNA,which are subsequently used to generate knockout mice. These mice may beemployed to study the zalpha11 gene and the protein encoded thereby inan in vivo system, and can be used as in vivo models for correspondinghuman diseases. Moreover, transgenic mice expression of zalpha11antisense polynucleotides or ribozymes directed against zalpha11,described herein, can be used analogously to transgenic mice describedabove.

[0178] For pharmaceutical use, the soluble receptor polypeptides of thepresent invention are formulated for parenteral, particularlyintravenous or subcutaneous, delivery according to conventional methods.Intravenous administration will be by bolus injection or infusion over atypical period of one to several hours. In general, pharmaceuticalformulations will include a zalpha11 soluble receptor polypeptide incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein ioss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, Gennaro,ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995. Therapeutic doseswill generally be in the range of 0.1 to 100 μg/kg of patient weight perday, preferably 0.5-20 mg/kg per day, with the exact dose determined bythe clinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins may be administered for acute treatment, over one week orless, often over a period of one to three days or may be used in chronictreatment, over several months or years. In general, a therapeuticallyeffective amount of zalpha11 soluble receptor polypeptide is an amountsufficient to produce a clinically significant effect.

[0179] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Identification of Human zalpha11 Using an ESTSequence to Obtain Full-length zalpha11

[0180] Scanning of a translated DNA database resulted in identificationof an expressed sequence tag (EST) sequence found to be a member of theClass I Cytokine Receptor family and designated zalpha11.

[0181] Confirmation of the EST sequence was made by sequence analyses ofthe cDNA from which the EST originated. This cDNA clone was obtained andsequenced using the following primers: ZC 447 (SEQ ID NO:5), ZC 976 (SEQID NO:6), ZC 19345 (SEQ ID NO:7), ZC 19346 (SEQ ID NO:8), ZC 19349 (SEQID NO:9), and ZC 19350 (SEQ ID NO:10), ZC 19458 (SEQ ID NO:11), ZC 19459(SEQ ID NO:12), ZC 19460 (SEQ ID NO:13), ZC 19461 (SEQ ID NO: 14), ZC19572 (SEQ ID NO:15), ZC 19573 (SEQ ID NO:16), ZC 19657 (SEQ ID NO: 17).The insert was 2945 bp, and was full-length.

Example 2 Tissue Distribution

[0182] Northern blot analysis was performed using Human Multiple TissueNorthern™ Blots (MTN I, MTN II, and MTN III) (Clontech). The cDNAdescribed in Example 1 was used in a PCR reaction using oligos ZC19,181(SEQ ID NO:18) and ZC19,182 (SEQ ID NO:19) as primers. PCR conditionswere as follows: 94° C. for 1.5 minutes; 35 cycles at 94° C. for 15seconds then 68° C. for 30 seconds; 72° C. for 10 minutes; 4° C.overnight A sample of the PCR reaction product was run on a 1.5% agarosegel. A band of the expected size of 175 bp was seen. The 175 bp PCRfragment, was gel purified using a commercially available kit (QiaexII™;Qiagen) and then radioactively labeled with ³²P-dCTP using Rediprime II™(Amersham), a random prime labeling system, according to themanufacturer's specifications. The probe was then purified using aNuc-Trap™ column (Stratagene) according to the manufacturer'sinstructions. ExpressHyb™ (Clontech) solution was used forprehybridization and as a hybridizing solution for the Northern blots.Hybridization took place overnight at 65° C. using 1-2×10⁶ cpm/ml oflabeled probe. The blots were then washed 4 times for 15 minutes in2×SSC/1% SDS at 25° C., followed by a wash in 0.1×SSC/0.1% SDS at 50° C.Transcripts of approximately 3 kb and 5 kb were detected in lymph node,peripheral blood leukocytes, and thymus.

[0183] Dot Blots were also performed using Human RNA Master Blots™(Clontech). The methods and conditions for the Dot Blots are the same asfor the Multiple Tissue Blots described above. Dot blot had strongestsignals in thymus, lymph node, and spleen.

[0184] Northern analysis was also performed using Human Cancer Cell LineMTN™ (Clontech). The cDNA described in Example 1 was used in a PCRreaction using oligos ZC19,907 (SEQ ID NO:20) and ZC19,908 (SEQ IDNO:21) as primers. PCR conditions were as follows: 35 cycles at 95° C.for 1 minute, then 60° C. for 1 minute; 72° C. for 1.5 minutes; 72° C.for 10 minutes; 4° C. overnight A sample of the PCR reaction product wasrun on a 1.5% agarose gel. A band of the expected size of 1.2 kb wasseen. The 1.2 kb PCR fragment, was gel purified using a commerciallyavailable kit (QiaQuick™ Gel Extraction Kit; Qiagen) and thenradioactively labeled with ³²P-dCTP using Prime-It II™ (Stratagene), arandom prime labeling system, according to the manufacturer'sspecifications. The probe was then purified using a Nuc-Trap™ column(Stratagene) according to the manufacturer's instructions. ExpressHyb™(Clontech) solution was used for prehybridization and as a hybridizingsolution for the Northern blots. Hybridization took place for 2 hours at65° C. using 1-2×10⁶ cpm/ml of labeled probe. The blots were then washed4 times for 15 minutes in 2×SSC/1% SDS at 25° C., followed by two 30minute washes in 0.1×SSC/0.1% SDS at 50° C. A strong signal was seen inthe Raji cell line derived from Burkitt's lymphoma.

Example 3 PCR-Based Chromosomal Mapping of the zalpha11 Gene

[0185] Zalpha11 was mapped to chromosome 16 using the commerciallyavailable “GeneBridge 4 Radiation Hybrid Panel” (Research Genetics,Inc., Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panelcontains PCRable DNAs from each of 93 radiation hybrid clones, plus twocontrol DNAs (the HFL donor and the A23 recipient). A publicly availableWWW server (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl)allows mapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map) which was constructed with the GeneBridge 4Radiation Hybrid Panel.

[0186] For the mapping of Zalpha11 with the “GeneBridge 4 RH Panel”, 20μl reactions were set up in a 96-well microtiter plate (Stratagene, LaJolla, Calif.) and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 95 PCR reactions consisted of 2 μl 10×PCRreaction buffer (Clontech Laboratories, Inc., Palo Alto, Calif.), 1.6 μldNTPs mix (2.5 mM each, Perkin-Elmer, Foster City, Calif.), 1 μl senseprimer, ZC 19,954, (SEQ ID NO:22), 1 μl antisense primer, ZC 19,955 (SEQID NO:23), 2 μl “RediLoad” (Research Genetics, Inc., Huntsville, Ala.),0.4 μl 50×Advantage KlenTaq Polymerase Mix (Clontech), 25 ng of DNA froman individual hybrid clone or control and ddH2O for a total volume of 20μl. The reactions were overlaid with an equal amount of mineral oil andsealed. The PCR cycler conditions were as follows: an initial 1 cycle 4minute denaturation at 94° C.; 35 cycles of a 45 seconds at 94° C., 45seconds at 68° C., and 1 minute at 72° C.; followed by 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel(Life Technologies).

[0187] The results showed that zalpha11 maps 9.54 cR_(—)3000 from theframework marker WI-3768 on the chromosome 16 WICGR radiation hybridmap. Proximal and distal framework markers were WI-3768 andTIGR-A002K05, respectively. The use of surrounding markers positionsZalpha11 in the 16p11.1 region on the integrated LDB chromosome 16 map(The Genetic Location Database, University of Southhampton, WWW server:http://cedar.genetics. soton.ac.uk/public_html/) .

Example 4 Construction of Human MPL-zalpha11 Polypeptide Chimera: MPLExtracellular and TM Domain Fused to the zalpha11 IntracellularSignaling Domain

[0188] The extracellular and transmembrane domains of the MPL receptorwere isolated from a plasmid containing the MPL receptor (PHZ1/MPLplasmid) using PCR with primers ZC17,212 (SEQ ID NO:24) and ZC19,914(SEQ ID NO:25). The reaction conditions were as follows: 95° C. for 1min.; 35 cycles at 95° C. for 1 min., 45° C. for 1 min., 72° C. for 2min.; followed by 72° C. at 10 min.; then a 10C soak. The PCR productwas run on a 1% low melting point agarose (Boerhinger Mannheim,Indianapolis, Ind.) and the approximately 1.5 kb MPL receptor fragmentisolated using Qiaquick™ gel extraction kit (Qiagen) as permanufacturer's instructions.

[0189] The intracellular domains of zalpha11 were isolated from aplasmid containing zalpha11 receptor cDNA using PCR with primersZC19,913 (SEQ ID NO:26) and ZC20,097 (SEQ ID NO:27). The polynucieotidesequence corresponding to the zalpha11 receptor coding sequence is shownin SEQ ID NO: 1 from nucleotide 69 to 1682. The reaction conditions wereas per above. The PCR product was run on a 1% low melting point agarose(Boerhinger Mannheim) and the approximately 900 bp zalpha11 fragmentisolated using Qiaquick gel extraction kit as per manufacturer'sinstructions.

[0190] Each of the isolated fragments described above were mixed at a1:1 volumetric ratio and used in a PCR reaction using ZC17,212 (SEQ IDNO:24) and ZC20,097 (SEQ ID NO:27) to create the MPL-zalpha11 chimera.The reaction conditions were as follows: 95° C. for 1 min.; 35 cycles at95° C. for 1 min., 55° C. for 1 min., 72° C. for 2 min.; followed by 72°C. at 10 min.; then a 10° C. soak. The entire PCR product was run on a1% low melting point agarose (Boehringer Mannheim) and the approximately2.4 kb MPL-zalpha11 chimera fragment isolated using Qiaquick gelextraction kit (Qiagen) as per manufacturer's instructions. TheMPL-zalpha11 chimera fragment was digested with EcoRI (BRL) and XbaI(Boerhinger Mannheim) as per manufacturer's instructions. The entiredigest was run on a 1% low melting point agarose (Boehringer Mannheim)and the cleaved MPL-zalpha11 chimera isolated using Qiaquick™ gelextraction kit (Qiagen) as per manufacturer's instructions. Theresultant cleaved MPL-zalpha11 chimera was inserted into an expressionvector as described below.

[0191] Recipient expression vector pZP-5N was digested with EcoRI (BRL)and HindIII (BRL) as per manufacturer's instructions, and gel purifiedas described above. This vector fragment was combined with the EcoRI andXbaI cleaved MPL-zalpha11 chimera isolated above and a XbaI/HindIIIlinker fragment in a ligation reaction. The ligation was run using T4Ligase (BRL), at 15° C. overnight. A sample of the ligation waselectroporated in to DH10B ElectroMAX™ electrocompetent E. coli cells(25 μF, 200 Ω, 2.3V). Transformants were plated on LB+Ampicillin platesand single colonies screened by PCR to check for the MPL-zalpha11chimera using ZC17,212 (SEQ ID NO:24) and ZC20,097 (SEQ ID NO:27) usingthe PCR conditions as described above.

[0192] Confirmation of the MPL-zalpha11 chimera sequence was made bysequence analyses using the following primers: ZC12,700 (SEQ ID NO:28),ZC5,020 (SEQ ID NO:29), ZC6,675 (SEQ ID NO:30), ZC7,727 (SEQ ID NO:31),ZC8,290 (SEQ ID NO:32), ZC19,572 (SEQ ID NO:15), ZC6,622 (SEQ ID NO:33),ZC7,736 (SEQ ID NO:34), and ZC9,273 (SEQ ID NO:35). The insert wasapproximately 2.4 bp, and was full-length.

Example 5 MPL-zalpha11 Chimera Based Proliferation in BAF3 Assay UsingAlamar Blue

[0193] A. Construction of BaF3 Cells Expressing MPL-zalpha11 Chimera

[0194] BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell linederived from murine bone marrow (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135,1986), was maintained in complete media (RPMI medium (JRH BioscienceInc., Lenexa, Kans.) supplemented with 10% heat-inactivated fetal calfserum, 2 ng/ml murine IL-3 (mIL-3) (R & D, Minneapolis, Min.), 2mML-glutaMax-1™ (Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSNantibiotics (GIBCO BRL)). Prior to electroporation, pZP-5N/MPL-zalpha11DNA (Example 4) was prepared and purified using a Qiagen Maxi Prep kit(Qiagen) as per manufacturer's instructions. BaF3 cells forelectroporation were washed once in RPMI media and then resuspended inRPMI media at a cell density of 10⁷ cells/ml. One ml of resuspended BaF3cells was mixed with 30 μg of the pZP-5N/MPL-zalpha11 plasmid DNA andtransferred to separate disposable electroporation chambers (GIBCO BRL).Following a 15 minute incubation at room temperature the cells weregiven two serial shocks (800 IFad/300 V.; 1180 IFad/300 V.) delivered byan electroporation apparatus (CELL-PORATOR™; GIBCO BRL). After a 5minute recovery time, the electroporated cells were transferred to 50 mlof complete media and placed in an incubator for 15-24 hours (37° C., 5%CO₂). The cells were then spun down and resuspended in 50 ml of completemedia containing GeneticinTM (Gibco) selection (500 μg/ml G418) in aT-162 flask to isolate the G418-resistant pool. Pools of the transfectedBaF3 cells, hereinafter called BaF3/MPL-zalpha11 cells, were assayed forsignaling capability as described below.

[0195] B. Testing the Signaling Capability of the BaF3/MPL-zalpha11Cells Using an Alamar Blue Proliferation Assay

[0196] BaF3/MPL-zalpha11 cells were spun down and washed in the completemedia, described above, but without mIL-3 (hereinafter referred to as“mIL-3 free media”). The cells were spun and washed 3 times to ensurethe removal of the mIL-3. Cells were then counted in a hemacytometer.Cells were plated in a 96-well format at 5000 cells per well in a volumeof 100 μl per well using the mIL-3 free media.

[0197] Proliferation of the BaF3/MPL-zalpha11 cells was assessed usingthrombopoietin (TPO) diluted with mIL-3 free media to 500 ng/ml, 250ng/ml, 125 ng/ml, 62 ng/ml, 30 ng/ml, 15 ng/ml, 7.5 ng/ml, 3.75 ng/ml,1.8 ng/ml, 0.9 ng/ml, 0.5 ng/ml and 0.25 ng/ml concentrations. 100 μl ofthe diluted TPO was added to the BaF3/MPL-zalpha11 cells. The totalassay volume is 200 μl. Negative controls were run in parallel usingmIL-3 free media only, without the addition of TPO. The assay plateswere incubated at 37° C., 5% CO₂ for 3 days at which time Alamar Blue(Accumed, Chicago, Ill.) was added at 20 μl /well. Alamar Blue gives afluourometric readout based on number of live cells, and is thus adirect measurement of cell proliferation in comparison to a negativecontrol. Plates were again incubated at 37° C., 5% CO₂ for 24 hours.Plates were read on the Fmax™ plate reader (Molecular Devices Sunnyvale,Calif.) using the SoftMax™ Pro program, at wavelengths 544 (Excitation)and 590 (Emmission).

[0198] Results confirmed the signaling capability of the intracellularportion of the zalpha11 receptor as the thrombopoietin inducedproliferation at approximately 10 fold over back ground at 62 ng/ml andgreater.

Example 6 Construction of Expression Vector Expressing Full-lengthzalpha11

[0199] The entire zalpha11 receptor was isolated from a plasmidcontaining zalpha11 receptor cDNA using PCR with primers ZC19,905 (SEQID NO:36) and ZC19,906 (SEQ ID NO:37). The reaction conditions were asfollows: 95° C. for 1 min; 35 cycles at 95° C. for 1 min, 55° C. for 1min, 72° C. for 2 min; followed by 72° C. at 10 min; then a 10° C. soak.The PCR product was run on a 1% low melting point agarose (BoerhingerMannheim) and the approximately 1.5 kb zalpha11 cDNA isolated usingQiaquick™ gel extraction kit (Qiagen) as per manufacturer'sinstructions.

[0200] The purified zalpha11 cDNA was digested with BamHI (BoerhingerMannheim) and EcoRI (BRL) as per manufacturer's instructions. The entiredigest was run on a 1% low melting point agarose (Boerhinger Mannheim)and purified the cleaved zalpha11 fragment using Qiaquick gel extractionkit as per manufacturer's instructions. The resultant cleaved zalpha11chimera was inserted into an expression vector as described below.

[0201] Recipient expression vector pZP-5N was digested with BamHI(Boerhinger Mannheim) and EcoRI (BRL) as per manufacturer'sinstructions, and gel purified as described above. This vector fragmentwas combined with the BamHI and EcoRI cleaved zalpha11 fragment isolatedabove in a ligation reaction. The ligation was run using T4 Ligase(BRL), at 15° C. overnight. A sample of the ligation was electroporatedin to DH10B electroMAX™ electrocompetent E. coli cells (25 μF, 200 Ω,2.3V). Transformants were plated on LB+Ampicillin plates and singlecolonies screened by PCR to check for the zalpha11 sequence usingZC19,905 (SEQ ID NO:36) and ZC19,906 (SEQ ID NO:37) using the PCRconditions as described above.

[0202] Confirmation of the MPL-zalpha11 sequence was made by sequenceanalyses using the following primers: ZC12,700 (SEQ ID NO:28), ZC5,020(SEQ ID NO:29), ZC20,114 (SEQ ID NO:38), ZC19,459 (SEQ ID NO:12),ZC19,954 (SEQ ID NO:39), and ZC20,116 (SEQ ID NO:40). The insert wasapproximately 1.6 kb, and was full-length.

Example 7 Zalpha11 Based Proliferation in BAF3 Assay Using Alamar Blue

[0203] A. Construction of BaF3 Cells Expressing zalpha11 Receptor

[0204] BaF3 cells expressing the full-length zalpha11 receptor wereconstructed as per Example 5A above, using 30 μg of the zalpha11expression vector, described in Example 6 above. The BaF3 cellsexpressing the zalpha11 receptor mRNA were designated as BaF3/zalpha11.These cells were used to screen for a zalpha11 activity as describedbelow in Examples 8 and 12.

Example 8 Screening for zalpha11 Activity Using BaF3/Zalpha11 CellsUsing an Alamar Blue Proliferation Assay

[0205] A. Monkey Primary Source Used to Test for Presence of zalpha11Activity

[0206] Conditioned media from primary monkey spleen cells was used totest for the presence of activity as described below. Monkey spleencells were activated with 5 ng/ml Phorbol-12-myristate-13-acetate (PMA)(Calbiochem, San Diego, Calif.), and 0.5 μg/ml IonomycinTM (Calbiochem)for 72 h. The supernatant from the stimulated monkey spleen cells wasused to assay proliferation of the BaF3/zalpha11 cells as describedbelow.

[0207] B. Screening for zalpha11 Activity Using BaF3/Zalpha11 CellsUsing an Alamar Blue Proliferation Assay

[0208] BaF3/Zalpha11 cells were spun down and washed in mIL-3 freemedia. The cells were spun and washed 3 times to ensure the removal ofthe mIL3. Cells were then counted in a hemacytometer. Cells were platedin a 96-well format at 5000 cells per well in a volume of 100 μl perwell using the mIL-3 free media.

[0209] Proliferation of the BaF3/Zalpha11 cells was assessed usingconditioned media from activated monkey spleen (see Example 8A, above)was diluted with mIL-3 free media to 50%, 25%, 12.5%, 6.25%, 3.125%,1.5%, 0.75% and 0.375% concentrations. 100 μl of the diluted conditionedmedia was added to the BaF3/Zalpha11 cells. The total assay volume is200 μl. The assay plates were incubated at 37° C., 5% CO₂ for 3 days atwhich time Alamar Blue (Accumed, Chicago, Ill.) was added at 20 μl/well.Plates were again incubated at 37° C., 5% CO₂ for 24 hours. Plates wereread on the Fmax™ plate reader (Molecular devices) as described above(Example 5).

[0210] Results confirmed the proliferative response of the BaF3/Zalpha11cells to a factor present in the activate monkey spleen conditionedmedia. The response, as measured, was approximately 4-fold overbackground at the 50% concentration. The BaF3 wild type cells did notproliferate in response to this factor, showing that this factor isspecific for the Zalpha11 receptor.

[0211] C. Human Primary Source Used to Isolate Zalpha11 Activity

[0212] 100 ml blood draws were taken from each of six donors. The bloodwas drawn using 10×10 ml vacutainer tubes containing heparin. Blood waspooled from six donors (600 ml), diluted 1:1 in PBS, and separated usinga Ficoll-Paque® PLUS (Pharmacia Biotech, Uppsala, Sweden). The isolatedprimary human cell yield after separation on the ficoll gradient was1.2×10⁹ cells.

[0213] Cells were suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA, 2 mMEDTA). 1.6 ml of cell suspension was removed and 0.4 ml CD3 microbeads(Miltenyi Biotec, Auburn, Calif.) added. The mixture was incubated for15 min. at 4° C. These cells labeled with CD3 beads were washed with 30ml MACS buffer, and then resuspended in 2 ml MACS buffer.

[0214] A VS+ column (Miltenyi) was prepared according to themanufacturer's instructions. The VS+ column was then placed in aVarioMACS™ magnetic field (Miltenyi). The column was equilibrated with 5ml MACS buffer. The isolated primary human cells were then applied tothe column. The CD3 negative cells were allowed to pass through. Thecolumn was rinsed with 9 ml (3×3 ml) MACS buffer. The column was thenremoved from the magnet and placed over a 15 ml falcon tube. CD3+ cellswere eluted by adding 5 ml MACS buffer to the column and bound cellsflushed out using the plunger provided by the manufacturer. Theincubation of the cells with the CD3 magnetic beads, washes, and VS+column steps (incubation through elution) above were repeated five moretimes. The resulting CD3+ fractions from the six column separations werepooled. The yield of CD3+ selected human T-cells were 3×10⁸ total cells.

[0215] A sample of the pooled CD3+ selected human T-cells was removedfor staining and sorting on a fluorescent antibody cell sorter (FACS) toassess their purity. The CD3+ selected human T-cells were 91% CD3+cells.

[0216] The CD3+ selected human T-cells were activated by incubating inRPMI+5% FBS+PMA 10 ng/ml and Ionomycin 0.5 μg/ml (Calbiochem) for 13hours 37° C. The supernatant from these activated CD3+ selected humanT-cells was tested for zalpha11 activity as described below.

[0217] D. Testing Supernatant from Activated CD3+ Selected Human T-cellsfor Zalpha11 Activity Using BaF3/Zalpha11 Cells and an Alamar BlueProliferation Assay

[0218] BaF3/Zalpha11 cells were spun down and washed in mIL-3 freemedia. The cells were spun and washed 3 times to ensure the removal ofthe mIL-3. Cells were then counted in a hemacytometer. Cells were platedin a 96-well format at 5000 cells per well in a volume of 100 μl perwell using the mIL-3 free media.

[0219] Proliferation of the BaF3/Zalpha11 cells was assessed usingconditioned media from activated CD3+ selected human T-cells (seeExample 8C, above) diluted with mIL-3 free media to 50%, 25%, 12.5%,6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations. 100 μl of thediluted conditioned media was added to the BaF3/Zalpha11 cells. Thetotal assay volume is 200 μl. The assay plates were incubated andassayed as described in Example 8B above.

[0220] Results confirmed the proliferative response of the BaF3/Zalpha11cells to a factor present in the activated CD3+ selected human T-cellconditioned media. The response, as measured, was approximately 10-foldover background at the 50% concentration. The BaF3 wild type cells didnot proliferate in response to this factor, showing that this factor isspecific for the Zalpha11 receptor.

Example 9 Construction of Mammalian Expression Vectors Tthat Expresszalpha11 Soluble Receptors: zalpha11CEE, zalpha11CFLG, zalpha11CHIS andzalph11-Fc4

[0221] A. Construction of zalpha11 Mammalian Expression VectorContaining zalphl ICEE, zalph11CFLG and zalph11CHIS

[0222] An expression vector was prepared for the expression of thesoluble, extracellular domain of the zalpha11 polypeptide,pC4zalph11CEE, wherein the construct is designed to express a zalpha11polypeptide comprised of the predicted initiating methionine andtruncated adjacent to the predicted transmembrane domain, and with aC-terminal Glu-Glu tag (SEQ ID NO:41).

[0223] A 700 bp PCR generated zalpha11 DNA fragment was created usingZC19,931 (SEQ ID NO:42) and ZC19,932 (SEQ ID NO:43) as PCR primers toadd Asp718 and BamHI restriction sites. A plasmid containing thezalpha11 receptor cDNA was used as a template. PCR amplification of thezalpha11 fragment was performed as follows: Twenty five cycles at 94Cfor 0.5 minutes; five cycles at 94° C. for 10 seconds, 50° C. for 30seconds, 68° C. for 45 seconds, followed by a 4° C. hold. The reactionwas purified by chloroform/phenol extraction and isopropanolprecipitation, and digested with Asp718 and BamHI (Gibco BRL) followingmanufacturer's protocol. A band of the predicted size, 700 bp, wasvisualized by 1% agarose gel electrophoresis, excised and the DNA waspurified using a QiaexII™ purification system (Qiagen) according themanufacturer's instructions.

[0224] The excised DNA was subcloned into plasmid pC4EE which had beencut with BamHI and Asp718. The pC4zalph11CEE expression vector uses thenative zalpha11 signal peptide and attaches the Glu-Glu tag (SEQ IDNO:41) to the C-terminus of the zalpha11 polypeptide-encodingpolynucleotide sequence. Plasmid pC4EE, is a mammalian expression vectorcontaining an expression cassette having the mouse metallothionein-1promoter, multiple restriction sites for insertion of coding sequences,a stop codon and a human growth hormone terminator. The plasmid also hasan E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene and the SV40 terminator.

[0225] About 30 ng of the restriction digested zalpha11 insert and about12 ng of the digested vector were ligated overnight at 16° C. Onemicroliter of each ligation reaction was independently electroporatedinto DH10B competent cells (GIBCO BRL, Gaithersburg, Md.) according tomanufacturer's direction and plated onto LB plates containing 50 mg/mlampicillin, and incubated overnight. Colonies were screened byrestriction analysis of DNA prepared from 2 ml liquid cultures ofindividual colonies. The insert sequence of positive clones was verifiedby sequence analysis. A large scale plasmid preparation was done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

[0226] The same process was used to prepare the zalpha11 solublereceptors with a C-terminal his tag, composed of 6 His residues in arow; and a C-terminal flag (SEQ ID NO:49) tag, zalpha11CFLAG. Toconstruct these constructs, the aforementioned vector has either the HISor the FLAG® tag in place of the glu-glu tag (SEQ ID NO:41).

[0227] B. Mammalian Expression Construction of Soluble zalpha11 Receptorzalpha11-Fc4

[0228] An expression plasmid containing all or part of a polynucleotideencoding zalpha11 was constructed via homologous recombination. Afragment of zalpha11 cDNA was isolated using PCR that includes thepolynucleotide sequence from extracellular domain of the zalha11receptor. The two primers used in the production of the zalpha11fragment were: (1) The primers for PCR each include from 5′ to 3′ end:40 bp of the vector flanking sequence (5′ of the insert) and 17 bpcorresponding to the 5′ end of the zalpha11 extracellular domain (SEQ IDNO:44); and (2) 40 bp of the 5′ end of the Fc4 polynucleotide sequence(SEQ ID NO:45) and 17 bp corresponding to the 3′ end of the zalpha11extracellular domain (SEQ ID NO:46). The fragment of Fc-4 for fusionwith the zalpha11 was generated by PCR in a similar fashion. The twoprimers used in the production of the Fc4 fragment were: (1) a 5′ primerconsisting of 40 bp of sequence from the 3′ end of zalpha11extracellular domain and 17 bp of the 5′ end of Fc4 (SEQ ID NO:47); and(2) a 3′ primer consisting of 40 bp of vector sequence (3′ of theinsert) and 17 bp of the 3′ end of Fc4 (SEQ ID NO:48).

[0229] PCR amplification of the each of the reactions described abovewas perforrned as follows: one cycle at 94° C. for 2 minutes;twenty-five cycles at 94° C. for 30 seconds, 60° C. for 30 seconds, 72°C. for 1 minute; one cycle at 72° C. for 5 minutes; followed by a 4° C.hold. Ten μl of the 100 μl PCR reaction was run on a 0.8% LMP agarosegel (Seaplaque GTG) with 1×TBE buffer for analysis. The remaining 90 μlof PCR reaction is precipitated with the addition of 5 μl 1 M 4NaCl and250 μl of absolute ethanol. The expression vector used was derived fromthe plasmid pCZR199 (deposited at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, and is designatedNo. 98668), and was cut with SmaI (BRL). The expression vector wasderived from the plasmid pCZR199, and is a mammalian expression vectorcontaining an expression cassette having the CMV immediate earlypromoter, a consensus intron from the variable region of mouseimmunoglobulin heavy chain locus, multiple restriction sites forinsertion of coding sequences, a stop codon and a human growth hormoneterminator. The expression vector also has an E. coli origin ofreplication, a mammalian selectable marker expression unit having anSV40 promoter, enhancer and origin of replication, a DHFR gene and theSV40 terminator. The expression vector used was constructed from pCZR199by the replacement of the metallothionein promoter with the CMVimmediate early promoter.

[0230] One hundred microliters of competent yeast cells (S. cerevisiae)were combined with 10 μl containing approximately 1 μg each of thezalpha11 and Fc4 inserts, and 100 ng of Smal (BRL) digested expressionvector and transferred to a 0.2 cm electroporation cuvette. Theyeast/DNA mixtures were electropulsed at 0.75 kV (5 kV/cm), “infinite”ohms, 25 μF. To each cuvette is added 600 μl of 1.2 M sorbitol and theyeast was plated in two 300 μl aliquots onto two URA-D plates andincubated at 30° C.

[0231] After about 48 hours, the Ura+ yeast transformants from a singleplate were resuspended in 1 ml H₂O and spun briefly to pellet the yeastcells. The cell pellet was resuspended in 1 ml of lysis buffer (2%Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Fivehundred microliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

[0232] Transformation of electrocompetent E. Coli cells (DH10B,GibcoBRL) is done with 0.5-2 ml yeast DNA prep and 40 ul of DH10B cells.The cells were electropulsed at 2.0 kV, 25 mF and 400 ohins. Followingelectroporation, 1 ml SOC (2% Bactoë Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mMMgSO4, 20 mM glucose) was plated in 250 μl aliquots on four LB AMPplates (LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/LAmpicillin).

[0233] Individual clones harboring the correct expression construct forzalpha11-Fc4 were identified by restriction digest to verify thepresence of the zalpha11-Fc4 insert and to confirm that the various DNAsequences have been joined correctly to one another. The insert ofpositive clones were subjected to sequence analysis. Larger scaleplasmid DNA is isolated using the Qiagen Maxi kit (Qiagen) according tomanufacturer's instructions.

Example 10 Transfection And Expression of Zalpha11 Soluble ReceptorPolypeptides

[0234] BHK 570 cells (ATCC No. CRL-10314), passage 27, were plated at1.2×10⁶ cells/well (6-well plate) in 800 μl of serum free (SF) DMEMmedia (DMEM, Gibco/BRL High Glucose) (Gibco BRL, Gaithersburg, Md.). Thecells were transfected with expression plasmids containingzalpha11CEE/CFLG/CHIS described above (see, Example 9), usingLipofectin™ (Gibco BRL), in serum free (SF) DMEM. Three micrograms ofzalpha11CEE/CFLG/CHIS each were separately diluted into 1.5 ml tubes toa total final volume of 100 μl SF DMEM. In separate tubes, 15 μ ofLipofectin™ (Gibco BRL) was mixed with 100 μl of SF DMEM. TheLipofectin™ mix was incubated at room temperature for 30-45 minutes thenthe DNA mix was added and allowed to incubate approximately 10-15minutes at room temperature.

[0235] The entire DNA: Lipofectin™ mixture was added to the plated cellsand distributed evenly over them. The cells were incubated at 37° C. forapproximately five hours, then transferred to separate 150 mm MAXIplates in a final volume of 30 ml DMEM/5% fetal bovine serum (FBS)(Hyclone, Logan, Utah). The plates were incubated at 37° C., 5% CO₂,overnight and the DNA: Lipofectin™ mixture was replaced with selectionmedia (5% FBS/DMEM with 1 μM methotrexate (MTX))the next day.

[0236] Approximately 10-12 days post-transfection, the plates werewashed with 10 ml SF DMEM. The wash media was aspirated and replacedwith 7.25 ml serum-free DMEM. Sterile Teflon meshes (Spectrum MedicalIndustries, Los Angeles, Calif.) pre-soaked in SF DMEM were then placedover the clonal cell colonies. A sterile nitrocellulose filterpre-soaked in SF DMEM was then placed over the mesh. Orientation markson the nitrocellulose were transferred to the culture dish. The plateswere then incubated for 5-6 hours in a 37° C., 5% CO₂ incubator.

[0237] Following incubation, the filters/meshes were removed, and themedia aspirated and replaced with 5% FBS/DMEM with 1 μM MTX. The filterswere then blocked in 10% nonfat dry milk/Western A buffer (Western A: 50mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and 0.25% gelatin)for 15 minutes at room temperature on a rotating shaker. The filterswere then incubated with an anti-Glu-Glu, anti-FLAG®, or anti-HISantibody-HRP conjugates, respectively, in 2.5% nonfat dry milk/Western Abuffer for one hour at room temperature on a rotating shaker. Thefilters were then washed three times at room temperature with Western Afor 5-10 minutes per wash. The filters were developed with ultra ECLreagent (Amersham Corp., Arlington Heights, Ill.) according themanufacturer's directions and visualized on the Lumi-Imager (RocheCorp.)

[0238] Positive expressing clonal colonies were mechanically picked to12-well plates in one ml of 5%FCS/DMEM with 5 μM MTX, then grown toconfluence. Conditioned media samples were then tested for expressionlevels via SDS-PAGE and Western anlaysis. The three highest expressingclones for each construct were picked; two out of three were frozen downas back up and one was expanded for mycoplasma testing and large-scalefactory seeding.

[0239] B. Mammalian Expression of soluble zalpha11 Receptor Zalpha11-Fc4

[0240] BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm tissueculture dishes and allowed to grow to approximately 50 to 70% confluencyovernight at 37_C, 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum (Hyclone,Logan, Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mMsodium pyruvate (Gibco BRL)). The cells were then transfected with theplasmid containing zalpha11-Fc4 (see, Example 9), using Lipofectamine™(Gibco BRL), in serum free (SF) media formulation (DMEM, 10 mg/mltransferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1%sodium pyruvate). The plasmid containing zalpha11-Fc4 was diluted into15 ml tubes to a total final volume of 640 ml with SF media. 35 ml ofLipofectamine™ (Gibco BRL) was mixed with 605 ml of SF medium. TheLipofectamine™ mix was added to the DNA mix and allowed to incubateapproximately 30 minutes at room temperature. Five milliliters of SFmedia was added to the DNA:Lipofectamine™ mixture. The cells were rinsedonce with 5 ml of SF media, aspirated, and the DNA:Lipofectamine™mixture is added. The cells were incubated at 37° C. for five hours,then 6.4 ml of DMEM/10% FBS, 1% PSN media was added to each plate. Theplates were incubated at 37° C. overnight and the DNA:Lipofectamine™mixture was replaced with fresh 5% FBS/DMEM media the next day. On day 2post-transfection, the cells were split into the selection media(DMEM/FBS media from above with the addition of 1 mM methotrexate (SigmaChemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50.The media on the cells was replaced with fresh selection media at day 5post-transfection. Approximately 10 days post-transfection, two 150 mmculture dishes of methotrexate resistant colonies from each transfectionwere trypsinized and the cells are pooled and plated into a T-162 flaskand transferred to large scale culture.

Example 11 Purification of zalpha11 Soluble Rreceptors from BHK 570Cells

[0241] A. Purification of zalpha11CEE Polypeptide from BHK 570

[0242] Unless otherwise noted, all operations were carried out at 4° C.The following procedure was used for purifying zalpha11 polypeptidecontaining C-terminal GluGlu (EE) tags. Thirty liters of cell factoryconditioned media was concentrated to 1.6 liters with an Amicon S10Y3spiral cartridge on a ProFlux A30. A Protease inhibitor solution wasadded to the concentrated 1.6 liters of cell factory conditioned mediafrom transfected BHK 570 cells (see, Example 10) to final concentrationsof 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim). Samples were removed for analysis and the bulkvolume was frozen at −80° C. until the purification was started. Totaltarget protein concentrations of the concentrated cell factoryconditioned media was determined via SDS-PAGE and Western blot analysiswith the anti-EE HRP conjugated antibody.

[0243] A 100 ml column of anti-EE G-Sepharose (prepared as describedbelow) was poured in a Waters AP-5, 5 cm×10 cm glass column. The columnwas flow packed and equilibrated on a BioCad Sprint (PerSeptiveBioSystems, Framingham, Mass.) with phosphate buffered saline (PBS) pH7.4. The concentrated cell factory conditioned media was thawed, 0.2micron sterile filtered, pH adjusted to 7.4, then loaded on the columnovernight with 1 ml/minute flow rate. The column was washed with 10column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4), thenplug eluted with 200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide(Anaspec, San Jose, Calif.) at 5 ml/minute. The EE peptide used has thesequence EYMPME (SEQ ID NO:41). The column was washed for 10 CVs withPBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The pH of theglycine-eluted column was adjusted to 7.0 with 2 CVs of 5×PBS, thenequilibrated in PBS (pH 7.4). Five ml fractions were collected over theentire elution chromatography and absorbance at 280 and 215 nM weremonitored; the pass through and wash pools were also saved and analyzed.The EE-polypeptide elution peak fractions were analyzed for the targetprotein via SDS-PAGE Silver staining and Western Blotting with theanti-EE HRP conjugated antibody. The polypeptide elution fractions ofinterest were pooled and concentrated from 60 ml to 5.0 ml using a10,000 Dalton molecular weight cutoff membrane spin concentrator(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

[0244] To separate zalpha11CEE from other co-purifying proteins, theconcentrated polypeptide elution pooled fractions were subjected to aPOROS HQ-50 (strong anion exchange resin from PerSeptive BioSystems,Framingham, Mass.) at pH 8.0. A 1.0×6.0 cm column was poured and flowpacked on a BioCad Sprint. The column was counter ion charged thenequibrated in 20 mM TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). Thesample was diluted 1:13 (to reduce the ionic strength of PBS) thenloaded on the Poros HQ column at 5 ml/minute. The column was washed for10 CVs with 20 mM Tris pH 8.0 then eluted with a 40 CV gradient of 20 mMTris/1 M sodium chloride (NaCI) at 10 ml/minute. 1.5 ml fractions werecollected over the entire chromatography and absorbance at 280 and 215nM were monitored. The elution peak fractions were analyzed via SDS-PAGESilver staining. Fractions of interest were pooled and concentrated to1.5-2 ml using a 10,000 Dalton molecular weight cutoff membrane spinconcentrator (Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

[0245] To separate zalpha11CEE polypeptide from free EE peptide and anycontaminating co-purifying proteins, the pooled concentrated fractionswere subjected to chromatography on a 1.5×90 cm Sephadex S200(Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBS at aflow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractions werecollected across the entire chromatography and the absorbance at 280 and215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified zalpha11CEE polypeptide.

[0246] This purified material was finally sujectd to a 4 ml ActiCleanEtox (Sterogene) column to remove any remaining endotoxins. The samplewas passed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

[0247] On Western blotted, Coomassie Blue and Silver stained SDS-PAGEgels, the zalphalICEE polypeptide was one major band of an apparentmolecular weight of 50,000 Daltons. The mobility of this band was thesame on reducing and non-reducing gels.

[0248] The protein concentration of the purified material was performedby BCA analysis (Pierce, Rockford, Ill.) and the protein was aliquoted,and stored at −80° C. according to our standard procedures. On IEF(isoelectric focusing) gels the protein runs with a PI of less than 4.5.The concentration of zalpha11CEE polypeptide was 1.0 mg/ml.

[0249] Purified zalpha11CEE polypeptide was prepared for injection intorabbits and sent to R & R Research and Development (Stanwood, Wash.) forantibody production. Rabbits were injected to produceanti-huzalpha11-CEE-BHK serum (Example 15, below).

[0250] To prepare anti-EE Sepharose, a 100 ml bed volume of proteinG-Sepharose (Pharmacia, Piscataway, N.J.) was washed 3 times with 100 mlof PBS containing 0.02% sodium azide using a 500 ml Nalgene 0.45 micronfilter-unit. The gel was washed with 6.0 volumes of 200 mMtriethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an equalvolume of EE antibody solution containing 900 mg of antibody was added.After an overnight incubation at 4° C., unbound antibody was removed bywashing the resin with 5 volumes of 200 mM TEA as described above. Theresin was resuspended in 2 volumes of TEA, transferred to a suitablecontainer, and dimethylpimilimidate-2HCI (Pierce, Rockford, Ill.)dissolved in TEA, was added to a final concentration of 36 mg/ml ofprotein G-Sepharose gel. The gel was rocked at room temperature for 45min and the liquid was removed using the filter unit as described above.Nonspecific sites on the gel were then blocked by incubating for 10 min.at room temperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA.The gel was then washed with 5 volumes of PBS containing 0.02% sodiumazide and stored in this solution at 4° C.

[0251] B. Purification of zalpha11CFLAG Polypeptide from BHK 570

[0252] Unless otherwise noted, all operations were carried out at 4° C.The following procedure was used for purifying zalpha11 polypeptidecontaining C-terminal FLAG® (FLG) (Sigma-Aldrich Colo.) tags. Thirtyliters of cell factory conditioned media was concentrated to 1.7 literswith an Amicon S10Y3 spiral catridge on a ProFlux A30. A Proteaseinhibitor solution was added to the 1.7 liters of concentrated cellfactory conditioned media from transfected BHK 570 cells (see, Example10) to final concentrations of 2.5 mM ethylenediaminetetraacetic acid(EDTA, Sigma Chemical Co. St. Louis, Mo.), 0.003 mM leupeptin(Boehringer-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin(Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). Sampleswere removed for analysis and the bulk volume was frozen at −80° C.until the purification was started. Total target protein concentrationsof the cell factory conditioned media was determined via SDS-PAGE andWestern blot analysis with the anti-FLAG® (Kodak) HRP conjugatedantibody. A 125 ml column of anti- FLAG® M2-Agarose affinity gel(Sigma-Aldrich Co.) was poured in a Waters AP-5, 5 cm×10 cm glasscolumn. The column was flow packed and equilibrated on a BioCad Sprint(PerSeptive BioSystems, Framingham, Mass.) with phosphate bufferedsaline (PBS) pH 7.4. The concentrated cell factory conditioned media wasthawed, 0.2 micron sterile filtered, pH adjusted to 7.4, then loaded onthe column overnight with 1 ml/minute flow rate. The column was washedwith 10 column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4),then plug eluted with 250 ml of PBS (pH 6.0) containing 0.5 mg/ml FLAG®(Sigma-Aldrich Co.) peptide at 5 ml/minute. The FLAG® peptide used hasthe sequence DYKDDDDK (SEQ ID NO:49). The column was washed for 10 CVswith PBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The pH of theglycine-eluted column was adjusted to 7.0 with 2 CVs of 5×PBS, thenequilibrated in PBS (pH 7.4). Five ml fractions were collected over theentire elution chromatography and absorbence at 280 and 215 nM weremonitored; the pass through and wash pools were also saved and analyzed.The FLAG®-polypeptide elution peak fractions were analyzed for thetarget protein via SDS-PAGE Silver staining and Western Blotting withthe anti-FLAG HRP conjugated antibody. The polypeptide elution fractionsof interest were pooled and concentrated from 80 ml to 12 ml using a10,000 Dalton molecular weight cutoff membrane spin concentrator(Millipore, Bedford, Mass.) according to the manufacturer'sinstructions.

[0253] To separate zalpha11CFLG from other co-purifying proteins, thepolypeptide elution pooled fractions were subjected to a POROS HQ-50(strong anion exchange resin from PerSeptive BioSystems, Framingham,Mass.) at pH 8.0. A 1.0×6.0 cm column was poured and flow packed on aBioCad Sprint. The column was counter ion charged then equilibrated in20 mM TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). The sample wasdiluted 1:13 (to reduce the ionic strength of PBS) then loaded on thePoros HQ-50 column at 5 ml/minute. The column was washed for 10 columnvolumes (CVs) with 20 mM Tris pet 8.0 then eluted with a 40 CV gradientof 20 mM Tris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 mlfractions were collected over the entire chromatography and absorbanceat 280 and 215 nM were monitored. The elution peak fractions wereanalyzed via SDS-PAGE Silver staining. Fractions of interest were pooledand concentrated to 1.5-2 ml using a 10,000 Dalton molecular weightcutoff membrane spin concentrator (Millipore, Bedford, Mass.) accordingto the manufacturer's instructions.

[0254] To separate zalpha11CFLG polypeptide from free FLAG® peptide andany contaminating co-purifying proteins, the pooled concentratedfractions were subjected to chromatography on a 1.5×90 cm Sephacryl S200(Pharmacia, Piscataway, N.J.) column equilibrated and loaded in PBS at aflow rate of 1.0 ml/min using a BioCad Sprint. 1.5 ml fractions werecollected across the entire chromatograohy and the absorbance at 280 and215 nM were monitored. The peak fractions were characterized viaSDS-PAGE Silver staining, and only the most pure fractions were pooled.This material represented purified zalpha11CFLG polypeptide.

[0255] This purified material was finally sujectd to a 4 ml ActiCleanEtox (Sterogene) column to remove any remaining endotoxins. The samplewas passed over the PBS equilibrated gravity column four times then thecolumn was washed with a single 3 ml volume of PBS, which was pooledwith the “cleaned” sample. The material was then 0.2 micron sterilefiltered and stored at −80° C. until it was aliquoted.

[0256] On Western blotted, Coomassie Blue and Silver stained SDS-PAGEgels, the zalpha11CFLG polypeptide was one major band of an apparentmolecular weight of 50,000 Daltons. The mobility of this band was thesame on reducing and non-reducing gels.

[0257] The protein concentration of the purified material was performedby BCA analysis (Pierce, Rockford, Ill.) and the protein was aliquoted,and stored at −80° C. according to our standard procedures. On IEF(isoelectric focusing) gels the protein runs with a PI of less than 4.5.The concentration of zalpha11CFLG polypeptide was 1.2 mg/ml.

[0258] C. Purification of zalpha11-Fc4 Polypeptide from Transfected BHK570 Cells

[0259] Unless otherwise noted, all operations were carried out at 4° C.The following procedure was used for purifying zalpha11 polypeptidecontaining C-terminal fusion to human IgG/Fc (zalpha11-Fc4; Examples 8and 9). 12,000 ml of conditioned media from BHK 570 cells transfectedwith zalpha11-Fc4 (Example 10) was filtered through a 0.2 mm sterilizingfilter and then supplemented with a solution of protease inhibitors, tofinal concentrations of, 0.001 mM leupeptin (Boerhinger-Mannheim,Indianapolis, Ind.), 0.001 mM pepstatin (Boerhinger-Mannheim) and 0.4 mMPefabloc (Boerhinger-Mannheim). A protein G sepharose (6 ml bed volume,Pharmacia Biotech) was packed and washed with 500 ml PBS (Gibco/BRL) Thesupplemented conditioned media was passed over the column with a flowrate of 10 ml/minute, follOowed by washing with 1000 ml PBS (BRL/Gibco).zalpha11-Fc4 was eluted from the column with 0.1 M Glycine pH 3.5 and 2ml fractions were collected directly into 0.2 ml 2M Tris pH 8.0, toadjust the final pH to 7.0 in the fractions.

[0260] The eluted fractions were characterized by SDS-PAGE and westernblotting with anti-human Fc (Amersham) antibodies. Western blot analysisof reducing SDS-PAGE gels reveal an immunoreactive protein of 80,000 KDain fractions 2-10. Silver stained SDS-PAGE gels also revealed an 80,000KDa zalpa11:Fc polypeptide in fractions 2-10. Fractions 2-10 werepooled.

[0261] The protein concentration of the pooled fractions was performedby BCA analysis (Pierce, Rockford, Ill.) and the material was aliquoted,and stored at −80° C. according to our standard procedures. Theconcentration of the pooled fractions was 0.26 mg/ml.

Example 12 Assay Using zalpha11 Soluble Receptor Zalpha11CEE,Zalpha11CFLG and zalpha11-Fc4 (Mutant) Soluble Receptors in CompetitiveInhibition Assay

[0262] BaF3/Zalpha11 cells were spun down and washed in mIL-3 freemedia. The cells were spun and washed 3 times to ensure the removal ofthe mIL-3. Cells were then counted in a hemacytometer. Cells were platedin a 96-well format at 5000 cells per well in a volume of 100 μl perwell using the mIL-3 free media.

[0263] Both media from the monkey spleen cell activation and the CD3+selected cells, described in Example 8 above, were added in separateexperiments at 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375%concentrations, with or without zalpha11 soluble receptors (CEE, C-flag,and Fc4 constructs; See, Example 10 and 11) at 10 μg/ml. The total assayvolume was 200 μl.

[0264] The assay plates were incubated 37° C., 5% CO₂ for 3 days atwhich time Alamar Blue (Accumed) was added at 20 μl/well. Plates wereagain incubated at 37° C., 5% CO₂ for 24 hours. Plates were read on theFmax™ plate reader (Molecular Devices) as described above (Example 5).Results demonstrated complete inhibition of cell growth from each of thedifferent zalpha11 soluble receptor constructs at 10 μg/ml, confirmingthat the factor in each sample was specific for the zalpha11 receptor.

[0265] Titration curves, diluting out the soluble receptors, were alsorun using the above stated assay. Both the zalpha11CEE and zalpha11CFLGsoluble zalpha11 receptors were able to completely inhibit growth as lowas 20 ng/ml. The mutant zalpha11-Fc4 soluble zalpha11 receptor was onlyas effective at 1.5 μg/ml.

Example 13 Expression of Human Zalpha11 in E. coli

[0266] A. Construction of Expression Vector pCZR225 That Expresseshuzalpha11/MBP-6H Fusion Polypeptide

[0267] An expression plasmid containing a polynucleotide encoding ahuman zalpha11 soluble receptor fused C-terminally to maltose bindingprotein (MBP) was constructed via homologous recombination. Thepolynucleotide sequence for the MBP-zalpha11 soluble receptor fusionpolypeptide is shown in SEQ ID NO:50, with the corresponding proteinsequence shown in SEQ ID NO:51. The fusion polypeptide, designatedhuzalpha11/MBP-6H, in Example 14, contains an MBP portion (amino acid 1(Met) to amino acid 388 (Ser) of SEQ ID NO:51) fused to the humanzalpha11 soluble receptor (amino acid 389 (Cys) to amino acid 606 (His)of SEQ ID NO:51). A fragment of human zalpha11 cDNA (SEQ ID NO:52) wasisolated using PCR. Two primers were used in the production of the humanzalpha11 fragment in a PCR reaction: (1) Primer ZC20,187 (SEQ ID NO:53),containing 40 bp of the vector flanking sequence and 25 bp correspondingto the amino terminuis of the human zalphal 1, and (2) primer ZC20,185(SEQ ID NO:54), containing 40 bp of the 3′ end corresponding to theflanking vector sequence and 25 bp corresponding to the carboxylterminus of the human zalpha11. The PCR Reaction conditions were asfollows: 25 cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and72° C. for 1 minute; followed by 4° C. soak, run in duplicate. Two μl ofthe 100 μl PCR reaction was run on a 1.0% agarose gel with 1×TBE bufferfor analysis, and the expected approximately 660 bp fragment was seen.The remaining 90 μl of PCR reaction was combined with the second PCRtube. precipitated with 400 μl of absolute ethanol. The precipitated DNAused for recombining into the Sma1 cut recipient vector pTAP98 toproduce the construct encoding the MBP-zalpha11 fusion, as describedbelow.

[0268] Plasmid pTAP98 was derived from the plasmids pRS316 and pMAL-c2.The plasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (HieterP. and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E.coli expression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP98 was-constructed usingyeast homologous recombination. 100 ng of EcoR1 cut pMAL-c2 wasrecombined with 1 μg Pvu1 cut pRS316, 1 μg linker, and 1 μg Sca1/EcoR1cut pRS316. The linker consisted of oligos ZC19,372 (SEQ ID NO:55) (100pmol): ZC19,351 (SEQ ID NO:56) (1 pmol): ZC19,352 (SEQ ID NO:57) (1pmol), and ZC19,371 (SEQ ID NO:58) (100 pmol) combined in a PCRreaction. PCR reaction conditions were as follows: 10 cycles of 94° C.for 30 seconds, 50° C. for 30 seconds, and 72° C. for 30 seconds;followed by 4° C. soak. PCR products were concentrated via 100% ethanolprecipitation.

[0269] One hundred microliters of competent yeast cells (S. cerevisiae)were combined with 10 μl of a mixture containing approximately 1 μg ofthe human zalpha11 receptor PCR product above, and 100 ng of SmaIdigested pTAP98 vector, and transferred to a 0.2 cm electroporationcuvette. The yeast/DNA mixture was electropulsed at 0.75 kV (5 kV/cm),infinite ohms, 25 μF. To each cuvette was added 600 μl of 1.2 M sorbitoland the yeast was then plated in two 300 μl aliquots onto two-URA Dplates and incubated at 30° C.

[0270] After about 48 hours, the Ura+ yeast transfonnants from a singleplate were resuspended in 1 ml H₂O and spun briefly to pellet the yeastcells. The cell pellet was resuspended in 1 ml of lysis buffer (2%Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Fivehundred microliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroformn,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendoif centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

[0271] Transformation of electrocompetent E. coli cells (MC1061,Casadaban et. al. J. Mol. Biol. 138, 179-207) was done with 1 μl yeastDNA prep and 40 μl of MC1061 cells. The cells were electropulsed at 2.0kV, 25 μF and 400 ohms. Following electroporation, 0.6 ml SOC (2% Bacto™Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mMNaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was plated inone aliquot on MM/CA+AMP 100 mg/L plates (Pryor and Leiting, ProteinExpression and Pruification 10:309-319, 1997).

[0272] Cells harboring the correct expression construct for humanzalpha11 receptor were identified by expression. Cells were grown inMM/CA with 100 μg/ml Ampicillin for two hours, shaking, at 37° C. 1 mlof the culture was induced with 1 mM IPTG. 2-4 hours later the 250 μl ofeach culture was mixed with 250 1 acid washed glass beads and 250 μlThorner buffer with 5% βME and dye (8M urea, 100 mM Tris pH7.0, 10%glycerol, 2 mM EDTA, 5% SDS). Samples were vortexed for one minute andheated to 65° C. for 10 minutes. 20 μwere loaded per lane on a 4%-12%PAGE gel (NOVEX). Gels were run in 1×MES buffer. The positive cloneswere designated pCZR225 and subjected to sequence analysis. Thepolynucleotide sequence of MBP-zalpha11 fusion is shown in SEQ ID NO:50.

[0273] B. Bacterial Expression of Human huzalpha11IMBP-6H FusionPolypeptide

[0274] One microliter of sequencing DNA was used to transform strainBL21. The cells were electropulsed at 2.0 kV, 25 μF and 400 ohms.Following electroporation, 0.6 ml MM/CA with 100 mg/L Ampicillin.

[0275] Cells were grown in MM/CA with 100 μg/ml Ampicillin for two hoursshaking, at 37° C. 1 ml of the culture was induced with 1 mM IPTG. 2-4hours later the 250 μl of each culture was mixed with 250 μl acid washedglass beads and 250 μl Thorner buffer with 5% βME and dye (8M urea, 100mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples were vortexedfor one minute and heated to 65° C. for 10 minutes. 20 μl were loadedper lane on a 4%-12% PAGE gel (NOVEX). Gels were run in 1×MES buffer.The positive clones were used to grow up for protein purification of thehuzalpha11/MBP-6H fusion protein (Example 14, below).

Example 14 Purification of huzalpha11/MBP-6H Soluble Receptor FromE.coli Fermentation

[0276] Unless otherwise noted, all operations were carried out at 4° C.The following procedure was used for purifying huzalpha11/MBP-6H solublereceptor polypeptide. E. coli cells containing the pCZR225 construct andexpressing huzalpha11/MBP-6H soluble receptor (Example 13) were grown upin SuperBroth II (12 g/L Casien, 24 g/L Yeast Extract, 11.4 g/Ldi-potassium phosphate, 1.7 g/L Mono-potassium phosphate; BectonDickenson, Cockeysville, Md.), and frozen in 0.5% glycerol. Twenty gramsof the frozen cells in SuperBroth II+Glycerol were used to purify theprotein. The frozen cells were thawed and diluted 1:10 in a proteaseinhibitor solution (Extraction buffer) prior to lysing the cells andreleasing the huzalphal 1/MBP-6H soluble receptor protein. The dilutedcells contained final concentrations of 20 mM Tris (J T Baker,Philipsburg, N.J.) 100 mM Sodium Chloride (NaCl, Mallinkrodt, Paris,Ky.), 0.5 mM pheynlmethylsulfonyl fluoride (PMSF, Sigma Chemical Co.,St. Louis, Mo.), 2 μg/ml Leupeptin (Fluka, Switzerland), and 2 μg/mlAprotinin (Sigma). A French Press cell breaking system (Constant SystemsLtd., Warwick, UK) with temperature of −7 to −10° C. and 30K PSI wasused to lyse the cells. The diluted cells were checked for breakage byA₆₀₀ readings before and after the French Press. The lysed cells werecentrifuged@18,000G for 45 minutes to remove the broken cell debris, andthe supernatant used to purify the protein. Total target proteinconcentrations of the supernatant was determined via BCA Protein Assay(Pierce, Rockford, Ill.), according to manufacturer's instructions.

[0277] A 25 ml column of Talon Metal Affinity resin (Clontech, PaloAlto, Calif.) (prepared as described below) was poured in a Bio-Rad, 2.5cm D×10 cm H glass column. The column was packed and equilibrated bygravity with 10 column volumes (CVs) of Talon Equilibration buffer (20mM Tris, 100 mM NaCl, pH 8.0). The supernatant was batch loaded to Talonmetal affinity resin and was rocked overnight. The resin was poured backinto the column and was washed with 10 CV's of Talon Equilibrationbuffer by gravity, then gravity eluted with 140 ml of Elution buffer(Talon Equilibration buffer+200 mM Imidazole-Fluka Chemical). The taloncolumn was cleaned with 5 CVs of 20 mM 2-(N-Morhpholino) ethanesulfonicacid pH 5.0 (MES, Sigma), 5 CVs of distilled H₂O, then stored in 20%Ethanol/0.1% Sodium Azide. Fourteen ml fractions were collected over theentire elution chromatography and the fractions were read withabsorbance at 280 and 320 nM and BCA protein assay; the pass through andwash pools were also saved and analyzed. The protein elution fractionsof interest were pooled and loaded straight to Amylose resin (NewEngland Biolabs, Beverly, Mass.).

[0278] To obtain more pure huzalpha11/MBP-6H polypeptide, the talonaffinity elution pooled fractions were subjected to Amylose resin(22mls) at pH 7.4. A 2.5 cm D×10 cm H Bio-Rad column was poured, packedand equilibrated in 10 CVs of Amylose equilibration buffer-20 mM Tris (JT Baker), 100 mM NaCl (Mallinkrodt), 1 mM PMSF (Sigma), 10 mMbeta-Mercaptoethanol (BME, ICN Biomedicals Inc., Aurora, Ohio) pH 7.4.The sample was loaded by gravity flow rate of 0.5 ml/min. The column waswashed for 10 CVs with Amylose equilibration buffer, then eluted with 2CV of Amylose equilibration buffer+10 mM Maltose (Fluka Biochemical,Switzerland) by gravity. 5 ml fractions were collected over the entirechromatography and absorbance at 280 and 320 nM were read. The Amylosecolumn was regenerated with I CV of distilled H₂O, 5 CVs of 0.l% (w/v)SDS (Sigma), 5 CVs of distilled H₂O, and then 5 CVs of Amyloseequilibration buffer.

[0279] Fractions of interest were pooled and dialyzed in a Slide-A-Lyzer(Pierce) with 4×4 L PBS pH 7.4 (Sigma) to remove low molecular weightcontaminants, buffer exchange and desalt. After the changes of PBS, thematerial harvested represented the purified huzalpha11/MBP-6Hpolypeptide. The purified huzalpha11/MBP-6H polypeptide was analyzed viaSDS-PAGE Coomassie staining and Western blot analysis with theanti-rabbit HRP conjugated antibody (Rockland, Gilbertsville, Pa.). Theconcentration of the huzalpha11/MBP-6H polypeptide was 1.92 mg/ml asdetermined by BCA analysis.

[0280] Purified huzalpha11/MBP-6H polypeptide was prepared for injectioninto rabbits and sent to R & R Research and Development (Stanwood,Wash.) for antibody production. Rabbits were injected to produce antianti-huzalpha11/MBP-6H serum (Example 15, below).

Example 15 zalpha11 Polyclonal Antibodies

[0281] Polyclonal antibodies were prepared by immunizing two female NewZealand white rabbits with the purified huzalpha11/MBP-6H polypeptide(Example 14), or the purified recombinant zalpha11CEE soluble receptor(Example 11A). Corresponding polyclonal antibodies were designatedrabbit anti-huzalpha11/MBP-6H and rabbit anti-huzalpha11-CEE-BHKrespectively. The rabbits were each given an initial intraperitoneal(IP) injection of 200 mg of purified protein in Complete Freund'sAdjuvant (Pierce, Rockford, Ill.) followed by booster IP injections of100 mg purified protein in Incomplete Freund's Adjuvant every threeweeks. Seven to ten days after the administration of the third boosterinjection, the animals were bled and the serum was collected. Therabbits were then boosted and bled every three weeks.

[0282] The zalpha11-specific polyclonal antibodies were affinitypurified from the rabbit serum using an CNBr-SEPHAROSE 4B protein column(Pharmacia LKB) that was prepared using 10 mg of the purifiedhuzalpha11/MBP-6H polypeptide (Example 14) per gram CNBr-SEPHAROSE,followed by 20× dialysis in PBS overnight. Zalpha11-specific antibodieswere characterized by an ELISA titer check using 1 mg/ml of theappropriate protein antigen as an antibody target. The lower limit ofdetection (LLD) of the rabbit anti-huzalpha11/MBP-6H affinity purifiedantibody is a dilution of 500 pg/ml. The LLD of the rabbitanti-huzalpha11-CEE-BHK affinity purified antibody is a dilution of 50pg/ml.

Example 16 Identification of Cells Expressing zalpha11 Receptor UsingRT-PCR

[0283] Specific human cell types were isolated and screened for zalpha11expression by RT-PCR. B-cells were isolated from fresh human tonsils bymechanical disruption through 100 μm nylon cell strainers (Falcon™;Bectin Dickenson, Franklin Lakes, N.J.). The B-cell suspensions wereenriched for CD19+ B-cells by positive 25 selection with VarioMACS VS+magnetic column and CD19 microbeads (Miltenyi Biotec, Auburn, Calif.) asper manufacturer's instructions. T-cells and monocytes were isolatedfrom human apheresed blood samples. CD3+ T-cells were purified by CD3microbead VarioMACS positive selection and monocytes were purified byVarioMACS negative selection columns (Miltenyi) as per manufacturer'sinstructions. Samples from each population were stained and analyzed byfluorescent antibody cell sorting (FACS) (Bectin Dickinson, San Jose,Calif.) analysis to determine the percent enrichment and resultingyields. CD19+ B-cells were approximately 96% purified CD3+ T-cells wereapproximately 95% purified, and monocytes were approximately 96%purified.

[0284] RNA was prepared, using a standard method in the art, from allthree cell types that were either resting or activated. RNA was isolatedfrom resting cells directly from the column preparations above. TheCD19+ and CD3+ cells were activated by culturing at 500,000 cells/ml inRPMI+10%FBS containing PMA 5 ng/ml (Calbiochem, La Jolla, Calif.) andIonomycin 0.5 ug/ml (Calbiochem) for 4 and 24 hours. The monocytes wereactivated by culturing in RPMI+10% FBS containing LPS 10 ng/ml (SigmaSt. Louis Mo.) and rhIFN-g 10 ng/ml (R&D, Minneapolis, Min.) for 24hours. Cells were harvested and washed in PBS. RNA was prepared from thecell pellets using RNeasy Midiprep™ Kit (Qiagen, Valencia, Calif.) asper manufacturer's instructions and first strand cDNA synthesis wasgenerated with Superscript II™ Kit (GIBCO BRL, Grand Island, N.Y.) asper manufacturers protocol.

[0285] Oligos ZC19907 (SEQ ID NO:20) and ZC19908 (SEQ ID NO:21) wereused in a PCR reaction to screen the above described samples for a 1.2kb fragment corresponding to zalpha11 message. PCR amplification wasperformed with Taq Polymerase (BRL Grand Island N.Y.), and conditions asfollows: 35 cycles of 95° C. for 1 min., 60° C. for I min., 72° C. for30 sec.; 1 cycle at 72° C. for 10 min.; and 4° C. soak. 10 ul of each 50μl reaction volume was run on a 2% agarose IXTAE gel to identifyresultant products. PCR products were scored as (−) for no product, (+)for band visible, (++) increased presence of band and (+++) being themost predominant band, with results shown in Table 5 below. TABLE 5 cDNASource Activation PCR Product CD19+ cells  0 hr resting +  4 hractivated ++ 24 hr activated +++ CD3+ cells  0 hr resting −  4 hractivated ++ 24 hr activated − monocytes  0 hr resting − 24 hr activated−

[0286] These results indicated that zalpha11 message is present inresting human CD19+ B-cells and increases with mitogenic activation. Italso appears to be expressed by human CD3+ T-cells only after 4 houractivation. There was no apparent message in either resting or activatedhuman monocytes.

Example 17 Zalpha11 Immunohistochemistry

[0287] A. Cell and Tissue Preparations

[0288] Positive control tissues consisted of BaF3 cells transfected withzalpha11 (Example 7) and lymphoid tissues known to express zalpha11including mouse lymph node, spleen and thymus received from HSD (HarlanSprague Dawley, Indianapolis, Ind.), monkey lymph node and spleenreceived from Regional Primate Research Center (University ofWashington, Seattle, Wash.), human lymph node and spleen received fromCHTN (Cleveland, Ohio). Negative controls performed on each tissuesample included: (1) untransfected BaF3 cells, (2) liver and brain frommouse and human known not to express zalpha11, (3) staining withantibody dilution buffer (Ventann Bioteck Systems, Tucson Ariz.) in theabsence of primary antibody, and (4) using zalpha11 soluble protein incompetition experiments.

[0289] Other cell samples were examined. Both non-stimulated andstimulated HL60 cells were assayed. HL60 cells are a promyelocytic cellline, which can be differentiated into myeloid or granulocyte lineageswith different reagents. Stimulated HL60 samples were prepared asfollows: (1) HL60 cells were treated with lOng/mI ofphorbol-myristate-acetate (PMA) (Sigma, St. Louis, Mo.) for 48 hours todifferentiate into monocyte lineage cells; and (2) HL60 cells treatedwith 1.25% DMSO (Sigma) for 4 days to differentiate into neutrophil-likecells. In addition, human polymorphonuclear (PMN) cells, humangranulocytes, human peripheral blood lymphocytes (PBL) and humanmonocytes from fresh human blood were examined (prepared in house usingroutine methods in the art). The cells and tissues described above werefixed overnight in 10% NBF (Surgipath, Richmond, Ill.), and embedded inparapalst X-tra (Oxford Scientific, St. Louis, Mo.), and sectioned at 5μm with a Reichart-Jung 2050 microme (Leica Instruments GmbH, Nussloch,Germany).

[0290] B. Immunohistochemistry

[0291] Tissue slides were deparaffinized, hydrated to buffer (water),and subjected to steam HIER treatment in Antigen Retrieval Citra buffer(BioGenex, San Roman, Calif.) for 20 minutes. 5% normal goat serum(Vector, Burlingame, Calif.) was used to block non-specific binding for10 minutes. Immunocytochemical screening analyses were performed usingpolyclonal antibodies to zalpha11 soluble receptor protein (rabbitanti-huzalpha11-MBP-6H and rabbit anti-huzalpha11-CEE-BHK; see, Example15) as the primary antibodies, at dilutions of 1:200 and 1:400respectively. Biotin conjugated goat anti-rabbit IgG (Vector; Cat. No.BA-1000, 1.5 mg/ml) was used as the secondary antibody at dilution of1:200. In separate samples, protein competition was performed by usingadditional zalpha11CEE soluble receptor protein (in 10× fold excess)(Example 11A) to the primary antibody to pre-block primary antibodyimmunoreaction. This competition was used as a control for the rabbitpolyclonal antibody specificity to zalpha11. Detection was performed onthe Ventana ChemMate 500 instrument using a ChemMate DAB Kit (labeledStreptavidin-Biotin Kit with application of a streptavidin-horseradishperoxidase conjugate, and DAB substrate) according to manufacturer'sinstruction and using the manufacturer's hematoxylin counterstain for 30seconds (Ventana Biotek Systems, Tucson, Ariz.).

[0292] High expression of zalpha11 was observed in the PMA-activatedBL60 cells. Low level expression was observed in PBL and HL60 cellswithout stimulation. A subset of cells in the spleen, thymus and lymphnode of mouse showed positive staining. Lymph node and spleen of bothhuman and monkey, and IL60 cells with DMSO stimulation showed minimal orno staining. The signal seen in the cells and tissues was mostlycompeted out by using the excess zalpha11 soluble receptor protein. Thenegative control tissues of brain and liver showed no staining.

Example 18 Identifying Peripheral Blood Mononuclear Cells (PBMNC's) ThatExpress zalpha11 Receptor Using Polyclonal Rabbit Anti-sera to zalpha11Soluble Receptor

[0293] 200 ml fresh heparinized blood was obtained from a normal donor.Blood was diluted 1:1 in PBS, and separated using a Ficoll-Paque PLUSgradient (Pharmacia Biotech Uppsala, Sweden), and the lymphocyteinterface collected. Cells were washed 2× in PBS and resuspended inRPMI+5% FBS media at a concentration of 2×10⁶ cells/ml.

[0294] In order to determine whether expression of zalpha11 receptor isaffected by the activation state of the lymphocyte cells, i.e., betweenresting and activated cells several stimulation conditions were used: 1)un-stimulated, i.e., media alone (RPMI+5% FBS media); 2) stimulated withPMA 10 ng/ml+Ionomycin 0.5 μg/ml (both from Calbiochem); and 3) PHAactivation (phytohemagglutinin-P, Difco/VWR). The cells were incubatedat 37° C. for 17 hours then collected for staining to detect expressionof zalpha11 receptor.

[0295] An indirect staining protocol was used. Briefly, the humanlymphocyte cells were suspended in staining buffer (PBS+0.02% NaN3 +BSA1% normal human serum 2%) and plated at 2×10⁵ cells in 50 μl/well in a96 well plate. Antibodies to the zalpha11CEE soluble receptor (Example15) were used to determine whether they co-stained with a B-cell(CD-19), T-cell (CD-3) or monocyte marker (CD-14) on the isolated humanlymphocytes. A rabbit polyclonal sera to zalpha11 soluble receptor (Rbanti-huzalpha11-CEE-BHK) (Example 15) at 10 μg/ml was used as theantibody to identify zalpha11 on the lymphocytes. A secondary antibody,goat anti-rabbit Ig-FITC (Biosource, Camarillo, Calif.), was used tovisualize the Rb anti-huzalpha11-CEE-BHK antibody binding to thezalpha11 receptors. Other antibodies were simultaneously used to stain Tcells (CD3-PE; PharMingen, San Diego, Calif.), B cells (CD19-PE)(PharMingen), and monocytes (CD-14-PE) (PharMingen) in order to identifyco-staining of the anti-zalpha11 receptor antibody on these cell types.Various controls were used to determine non-specific binding andbackground levels of staining: (1) an irrelevant rabbit polyclonal serawas used as a non-specific control; and (2) secondary antibody alone wasused to determine background binding of that reagent. Purified,zalpha11CEE soluble receptor (Example 11) was used in about a 10-foldexcess as a competitive inhibitor to verify the specificity of therabbit anti-huzalpha11-CEE-BHK antibody to zalpha11 soluble receptor.

[0296] After plating the cells and adding the primary and co-stainingantibodies, the cells were incubated on ice for 30 minutes, washed 2×with staining buffer, and stained with the secondary antibody, goatanti-rabbit Ig-FITC (Biosource), for 30 minutes on ice. Cells werewashed 2× staining buffer, and resuspended at 200 μl per well instaining buffer containing the viability stain 7AAD at about 1 μg/mlfinal concentration (Sigma, St. Louis, Mo.). Samples were read on theFACS-Caliber (Becton-Dickinson, San Jose, Calif.) and viable cellsanalyzed.

[0297] The rabbit polyclonal to zalpha11 receptor stained resting Bcells. The signal on resting B cells was brighter than the signalachieved using the irrelevant rabbit sera, and the signal was diminishedto a greater extent on B cells than on T cells with the addition ofexcess zalpha11-CEE soluble receptor. This experiment was repeated usingseparated B and T cells, and the results were very similar. Again thestaining with the polyclonal rabbit anti-huzalpha11-CEE-BHK antibody tozalpha11 receptor was highest on resting B cells.

Example 19 Zalpha11 Receptor Expression in Various Tissues UsingReal-Time Quantitative RT/PCR

[0298] A. Primers and Probes for Quantitative RT-PCR-

[0299] Real-time quantitative RT-PCR using the ABI PRISM 7700 SequenceDetection System (PE Applied Biosystems, Inc., Foster City, Calif.) hasbeen previously described (See, Heid, C. A. et al., Genome Research6:986-994, 1996; Gibson, U. E. M. et al., Genome Research 6:995-1001,1996; Sundaresan, S. et al., Endocrinology 139:4756-4764, 1998. Thismethod incorporates use of a gene specific probe containing bothreporter and quencher fluorescent dyes. When the probe is intact thereporter dye emission is negated due to the close proximity of thequencher dye. During PCR extension using additional gene-specificforward and reverse primers, the probe is cleaved by 5′ nucleaseactivity of Taq polymerase which releases the reporter dye from theprobe resulting in an increase in fluorescent emission.

[0300] The primers and probes used for real-time quantitative RT-PCRanalyses of zalpha11 expression were designed using the primer designsoftware Primer Express™ (PE Applied Biosystems, Foster City, Calif.).Primers for human zalpha11 were designed spanning an intron-exonjunction to eliminate amplification of genomic DNA. The forward primer,ZC22,277 (SEQ ID NO:59) and the reverse primer, ZC22,276 (SEQ ID NO:60)were used in a PCR reaction (below) at about 300 nM concentration tosynthesize a 143 bp product. The corresponding zalpha11 TaqMan® probe,designated ZG31 (SEQ ID NO:61) was synthesized and labeled by PE AppliedBiosystems. The ZG31 probe was labeled at the 5′ end with a reporterfluorescent dye (6-carboxy-fluorescein) (FAM) (PE Applied Biosystems)and at the 3′ end with a quencher fluorescent dye(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems).

[0301] As a control to test the integrity and quality of RNA samplestested, all RNA samples (below) were screened for rRNA using a primerand probe set ordered from PE Applied Biosystems (cat No. 4304483). Thekit contains an rRNA forward primer (SEQ ID NO:66) and the rRNA reverseprimer (SEQ ID NO:67), rRNA TaqMan® probe (SEQ ID NO:68) The rRNA probewas labeled at the 5′end with a reporter fluorescent dye VIC (PE AppliedBiosystems) and at the 3′ end with the quencher fluorescent dye TAMRA(PE Applied Biosystems). The rRNA results also serve as an endogenouscontrol and allow for the normalization of the zalpha11 mRNA expressionresults seen in the test samples.

[0302] RNA samples from human CD3, CD19 and monocyte cell types wereprepared and described as per Example 16 above. Control RNA wasprepared, using RNeasy Miniprep™ Kit (Qiagen, Valencia, Calif.) as permanufacturer's instructions, from approximately 10 million BaF3 cellsexpressing human zalpha11 receptor (Example 7).

[0303] B. Real-Time Quantitative RT-PCR-

[0304] Relative levels of zalpha11 mRNA were determined by analyzingtotal RNA samples using the one-step RT-PCR method (PE AppliedBiosystems). Total RNA from BaF3 cells expressing human zalpha11receptor was isolated by standard methods and used to generate astandard curve used for quantitation. The curve consisted of 10-foldserial dilutions ranging from 2.5-2.5×10⁻⁴ ng/μl for the rRNA screen and250-0.025 ng/μl for the zalpha11 screen with each standard curve pointanalyzed in triplicate. The total RNA samples from the human CD3, CD19and monocyte cells were also analyzed in triplicate for human zalpha11transcript levels and for levels of rRNA as an endogenous control. In atotal volume of 25 μl, each RNA sample was subjected to a One-StepRT-PCR reaction containing: approximately 25 ng of total RNA in buffer A(50 mM KCL, 10 mM Tris-HCL); the internal standard dye,carboxy-x-rhodamine (ROX)); appropriate primers (approximately 50 nMrRNA primers (SEQ ID NO:66 and SEQ ID NO:67) for the rRNA samples; andapproximately 300 nM ZC22,277 (SEQ ID NO:59) and ZC22,276 (SEQ ID NO:60)primers for zalpha11 samples); the appropriate probe (approximately 50nM rRNA TaqMan® probe (SEQ ID NO:68) for rRNA samples, approximately 100nM ZG31 (SEQ ID NO:61) probe for zalpha11 samples); 5.5 mM MgCl₂; 300 μMeach d-CTP, d-ATP, and d-GTP and, 600 μM of d-UTP; MuLV reversetranscriptase (0.25 U/μl); AmpliTaq™ Gold DNA polymerase (0.025 /μl) (PEApplied Biosystems); and RNase Inhibitor (0.4 U/μl) (PE AppliedBiosystems). PCR thermal cycling conditions were as follows: an initialreverse transcription (RT) step of one cycle at 48° C. for 30 minutes;followed by an AmpliTaq Gold™ (PE Applied Biosystems) activation step ofone cycle at 95° C. for 10 minutes; followed by 40 cycles ofamplification at 95° C. for 15 seconds and 60° C. for 1 minute.

[0305] Relative zalpha11 RNA levels were determined by using theStandard Curve Method as described by the manufacturer, PE Biosystems(User Bulletin No.2: ABI Prism 7700 Sequence Detection System, RelativeQuantitation of Gene Expression, Dec. 11, 1997). The rRNA measurementswere used to normalize the zalpha11 levels and the resting CD3+ RNAsample was used as a calibrator. Resting CD3 was arbitrarily chosen asthe calibrator and given a value of 1.00. The rest of the samples werecompared relative to the calibrator. Data are shown in Table 6 below.TABLE 6 Sample Resting 4 hr Stimulation 24 hr Stimulation CD3 1.00 15.2716.70 CD19 20.14 65.08 25.42 Monocytes 0.05 no data 0.26

[0306] There was a 15-fold increase in zalpha11 receptor expression inCD3+ at 4 and 24 hrs. Resting CD19 had 20 fold increase in receptorexpression relative to resting CD3+. There was a 3 fold increase with 4hr stimulation that fell back to resting levels by 24 hrs. Monocytesshowed no detectable zalpha11 receptor expression in this assay.

Example 20 Identification of Cells Expressing zalpha11 Receptor Using insitu Hybridization

[0307] Specific human tissues were isolated and screened for zalpha11expression by in situ hybridization. Various human tissues prepared,sectioned and subjected to in situ hybridization included thymus,spleen, tonsil, lymph node and lung. The tissues were fixed in 10%buffered formalin and blocked in paraffin using standard techniques(Example 17). Tissues were sectioned at 4 to 8 microns. Tissues wereprepared using a standard protocol (“Development of non-isotopic in situhybridization” at http://dir.niehs.nih.gov/dirlep/ish.html). Briefly,tissue sections were deparaffinized with HistoClear (NationalDiagnostics, Atlanta, Ga.) and then dehydrated with ethanol. Next theywere digested with Proteinase K (50 μg/ml) (Boehringer Diagnostics,Indianapolis, Ind.) at 37° C. for 2 to 20 minutes. This step wasfollowed by acetylation and re-hydration of the tissues.

[0308] Two in situ probes generated by PCR were designed against thehuman zalpha11 sequence. Two sets of oligos were designed to generateprobes for separate regions of the zalpha11 cDNA: (1) Oligos ZC23,684(SEQ ID NO:62) and ZC23,656 (SEQ ID NO:63) were used to generate a 413bp probe for zalpha11; and (2) Oligos ZC23,685 (SEQ ID NO:64) andZC23,657 (SEQ ID NO:65) were used to generate a 430 bp probe forzalpha11. The second probe is 1500 bp 3′ of the first zalpha11 probe.The antisense oligo from each set also contained the working sequencefor the T7 RNA polymerase promoter to allow for easy transcription ofantisense RNA probes from these PCR products. The PCR reactionconditions were as follows: 30 cycles at 94° C. for 30 sec, 60° C. for 1min., 72° C. for 1.5 min. The PCR products were purified by Qiagen spincolumns followed by phenol/chloroform extraction and ethanolprecipitation. Probes were subsequently labeled with digoxigenin(Boehringer) or biotin (Boehringer) using an In Vitro transcriptionSystem (Promega, Madison, Wis.) as per manufacturer's instruction.

[0309] In situ hybridization was performed with a digoxigenin- orbiotin-labeled zalpha11 probe (above). The probe was added to the slidesat a concentration of 1 to 5 pmol/ml for 12 to 16 hours at 55-60° C.Slides were subsequently washed in 2×SSC and 0.1×SSC at 50° C. Thesignals were amplified using tyramide signal amplification (TSA) (TSA,in situ indirect kit; NEN) and visualized with Vector Red substrate kit(Vector Lab) as per manufacturer's instructions. The slides were thencounter-stained with hematoxylin (Vector Laboratories, Burlingame,Calif.).

[0310] A signal was seen in the thymus, tonsil, lung, and lymph node.The positive-staining cells appeared to be lymphocytes and relatedcells.

Example 21 Isolation of the Mouse Zalpha11 Receptor

[0311] A. Mouse Genomic Library Screen

[0312] An initial partial mouse zalpha11 sequence was obtained byprobing a mouse genomic library with a human zalpha11 receptorpolynucleotide probe containing the entire cDNA. The human zalpha11 cDNAwas generated by PCR with ZC19,905 (SEQ ID NO:36) and ZC19,906 (SEQ IDNO:37) primers and a plasmid containing full length human zalpha11(e.g., Example 1) was used for the template. The PCR reaction conditionswere as follows: 35 cycles at 98° C for 1 min., 68° C. for 1 min., and72° C. for 2 min.; followed by one cycle at 72° C. for 10 min. The PCRproduct was run on a 1% low melting point agarose (Boerhinger Mannheim)and the approximately 1.5 kb human zalpha11 cDNA isolated usingQiaquick™ gel extraction kit (Qiagen) as per manufacturer'sinstructions. This human zalpha11 cDNA was used to screen a mousegenomic DNA library (below).

[0313] The mouse genomic DNA library used was emb13 SP6/T7 lambda BamHIcloned library (Clontech, Palo Alto, Calif.). This library representing7.2×10⁵ pfu was plated onto an E. coli K802 host lawn on 24 NZY plates.Plaque lifts were performned using Hybond-N filters (Amersham Pharmacia,Buckinghamshire, England, UK) as per manufacturer's instructions. Thefilters were denatured in 1.5 M NaCl and 0.5 M NaOH for 10 min. and thenneutralized in 1.5 M NaCl and 0.5 M Tris-HCL (pH 7.2 ) for 10 min. TheDNA was affixed to the filter using a STRATALINKER UV crosslinker(Stratagene) at 1200 joules. The filters were pre-washed to remove celldebris at 65° C. in pre-wash buffer (0.25×SSC, 0.25% SDS and 1 mM EDTA),changing solution three times for a total of 45 min. The filters wereprehybridized overnight at 50° C. in Expresshyb™ solution (Clontech)containing 0.1 mg/ml denatured salmon sperm DNA. Approximately 50 ng ofthe purified human zalpha11 cDNA (above) was labeled with ³²P using theRediprime II Random Prime Labeling System (Amersham Pharmacia) as permanufacturers instructions. Unincorporated radioactivity was removedfrom the zaplha11 cDNA probe using a NucTrap™ push column (Stratagene,La Jolla, Calif.). Filters were hybridized in Expresshyb™ solution(Clontech) containing about 0.5 to about 1×10⁶ cpm/ml zalpha11 cDNAprobe, about 0.1 mg/ml denatured salmon sperm DNA and denatured 0.5μg/ml cot-1 DNA. Hybridization took place overnight at 50° C. Filterswere washed in 2×SSC, 0.1% SDS at room temperature for 2 hours (changingwash several times) then the temperature was raised to 60° C. for onehour (changing buffer once). Overnight exposure at −80° C. showed 6plaques representing primary isolates.

[0314] To obtain secondary plaque isolates, the 6 plaques representingprimary isolates were picked with a Pasteur pipette and eluted overnightat 4° C. in 1 ml SM (0.1 M NaCl, 50 mM Tris pH 7.5, 10 mM MgSO₄, 0.02%gelatin) containing a few drops of chloroform. After determining phagetiters, about 12.5× the estimated amount of phage in the original plug(12.5× coverage) of 6 primary isolates was plated on a lawn of E. coliK802 cells embedded in 10 mM MgSO₄/NZY top agarose on NZY maxi plates,and grown overnight at 37° C. Plaque lifts were done using Hybond-Nfilters (Amersham Pharmacia) as per manufacturer's instructions. Filterswere fixed as per above. The second round filters were pre-washed toremove cell debris at 65° C. in pre-wash buffer (2×SSC, 0.1% SDS and 1mM EDTA), changing solution three times for a total of 45 min. Thesecond round filter lifts were then prehybridized, and the zalpha11 cDNAprobe prepared as described above.

[0315] The second round filters were hybridized as above in Expresshyb™solution (Clontech) containing about 10⁶ cpm/ml zalpha11 cDNA probecontaining about 0.1 mg/ml denatured salmon sperm DNA. Hybridizationtook place overnight at 50° C. Wash conditions described above for theprimary screen were repeated for this secondary screen. After anovernight exposure at −80° C., two of the 6 original primary plaquesisolates were verified as positive in the secondary screen. Positiveplaques hybridizing to human zalpha11 cDNA in the secondary screen werepicked with a Pasteur pipette and designated 7b1 and 20b1.

[0316] The isolated plaques No.7b1 and 20b1 were eluted in 200 μl SMovernight at 4 ° C. Serial 10-fold serial dilutions ranging from 10⁻² to10⁻⁶ of each isolate were plated on host E. coli K802 cells to determinethe titer. Isolate 20b1 had a titer of 4×10³ pfu/μl and was furtherpursued. 4 plates were prepared by plating 10⁵ pfu/plate on confluenthost E. coli K802 cells in order to make a phage DNA prep. Plates weregrown at 37° C. for about 6.5 hours until phage lysis was starting toget confluent. The phage was then eluted overnight at 4° C. in 12 ml ofSM per plate. Plates were then shaken at room temperature one hour, thesupernatant was removed; 1% chloroform was added and supernatant wasshaken again for 15 min. The 20b1 phage DNA was prepped using the WizardLambda Preps DNA Purification System (Promega, Madison, Wis.; sectionsIV and VI).

[0317] Samples of 20b1 phage DNA were cut with several restrictionenzymes to generate DNA fragments for Southern blotting. The digestswere run on a 1% TBE agarose gel. The gel was soaked in 0.25 M HCl for30 min.; rinsed in distilled H2O; soaked in 0.5M NaOH and 1.5 M NaCl for40 min. with one solution change and neutralized in 1.5 NaCl and 0.5Tris-HCL (pH 7.2) for 40 min. with one solution change. A TURBOBLOTTERTMRapid Downward Transfer System (Schleicher & Schuell, Keene, N.H.) wasset up to transfer the DNA onto a Nytran/BA-S membrane (Schleicher &Schuell) overnight. The DNA was affixed to the Nytran using aSTRATALINKER UV crosslinker (Stratagene, La Jolla, Calif.) at 1200joules. The blot was prehybridized as described above. About 50 ng ofthe human zalpha11 cDNA was labeled and purified for a probe, asdescribed above. Filters were hybridized as above in Expresshyb™solution (Clontech) containing about 10⁶ cpm/ml zalpha11 cDNA probe andabout 0.1 mg/ml denatured salmon sperm DNA. Hybridization took placeovernight at 50° C. The blot was washed as described above and exposedto film overnight at −80° C.

[0318] The Southern showed a DNA fragment generated from a BamHI/StuIdigest which hybridized to the human zalpha11 cDNA probe in the expectedsize range of 1.3 to 1.6 kb. This fragment was pursued. Approximately 3μg of 20b1 lambda DNA was cut with 20 units of BamHI (BoehringerMannheim, Indianapolis, Ind.) and 20 Units StuI (NEB, Beverly, Mass.)for 2 hours at 37° C. The digest was run on a 1% TBE gel and a 1.3 kbdoublet and 1.6 kb doublet bands were excised from the gel and the DNAwas extracted from the agarose using the Qiaquick Gel Extraction Kit(Qiagen, Valencia, Calif.). Due to the low yield of DNA from the prep,it was not possible to determine by additional restriction digestanalysis whether fragments which hybridized to the human zalpha11 cDNAprobe were BamHI/StuI or StuI/StuI fragments. Thus, blunt ligationsusing 5 μl of the 1.3 kb doublet fragment and 5 μl of the 1.6 kb doubletfragment were done using the Zero Blunt PCR Cloning Kit (Invitrogen,Carlsbad, Calif.). The blunt ligation yielded positive clones with bothof the 1.6 kb fragments and one of the 1.3 kb fragments. These cloneswere digested with EcoRI (Life Technologies) which flanks the T-overhangsite where the 1.6 and 1.3 kb fragments were inserted. Another Southernblot was performed to determine which was the original fragmenthybridizing to the human zalpha11 cDNA probe. The 1% TBE gel was treatedand the DNA was transferred to the Nytran blot as described above.

[0319] The blot was prehybridized as above in 10 ml of hybridizationsolution. A different human zalpha11 polynucleotide probe was prepared.Another full length zalpha11 cDNA human zalpha11 fragment was generatedfor use as a probe by PCR with the oligos ZC19,905 (SEQ ID NO: 36) andZC20,097 (SEQ ID NO: 27). The PCR reaction conditions were as follows:95° C. for 1 min.; 35 cycles of 95° C for 1 min., 55° C. for 1 min., and72° C. for 2 min.; followed by one cycle at 72° C. for 10 min. The PCRproduct was run on a 1% low melting point agarose (Boerhinger Mannheim)and the approximately 1.5 kb human zalpha11 cDNA isolated usingQiaquick™ gel extraction kit (Qiagen) as per manufacturer'sinstructions. About 50 ng of this isolated human zalpha11 cDNA fragmentwas labeled with ³²P and purified as described above. Filters werehybridized as above in Expresshyb™ solution (Clontech) containing about10⁶ cpm/ml zalpha11 cDNA probe, about 0.1 mg/ml denatured salmon sperm,and denatured 0.5 μg/ml cot-1 DNA. Hybridization and washing was asdescribed above. The blot was exposed to film 1.5 hours at −80° C. andthe 1.3 kb insert was strongly hybridizing to the human zalpha11 probe.

[0320] This clone was sequenced and found to contain a mouse zalpha11 3′coding exon with a termination codon and upstream intron sequence.Sequencing primers used were: ZC3,424 (SEQ ID NO:86), ZC694 (SEQ IDNO:87), ZC24,399 (SEQ ID NO:88), and ZC24,400 (SEQ ID NO:89). Thegenomic sequence of mouse zalpha11 including the 3′ exon is shown in SEQID NO:69. The the 3′ exon coding sequence starts at nucleotide 543 andends at nucleotide 1262 in SEQ ID NO:69, encoding a 240 amino acid exon(SEQ ID NO:70).

[0321] B. PCR Screen of Mouse cDNA Panel

[0322] A panel of available in-house and commercial mouse cDNAs(Clontech; Life technologies, Gaithersburg, Md.) was screened by PCRusing ZC24,432 (SEQ ID NO:71) and ZC24,433 (SEQ ID NO:72) as primers(about 20 pmol each). The PCR reaction conditions were as follows: 94°C., 2 min.; 32 cycles of 94° C. for 20 sec., 64° C. for 30 sec., and 72°C. for 30 sec.; followed by one cycle at 72° C for 5 min. Mouse spleen,dendritic cells, neonatal skin, bone marrow, wild type BaF3 cells, EL4cells, and lung showed strong PCR products of the predicted 450 bp size.

[0323] C. 5′ Nested RACE

[0324] 5′ RACE reactions were performed using 20 pmol each of primersZC9,739 (SEQ ID NO:73) and ZC24,434 (SEQ ID NO:74) and CD90+ selectedmouse spleen marathon cDNA as a template. The marathon cDNA was preparedusing a Marathon cDNA Amplification Kit (Clontech) according to themanufacturer's instructions. The PCR reaction conditions were asfollows: 94° C. for 1 min.; 5 cycles of 94° C. for 20 sec., and 70° C.for 1.5 min.; followed by 25 cycles of 94° C. for 20 sec., 64° C. for 20sec., and 70° C. for 1.5 min.; followed by one cycle at 72° C. for 5min.

[0325] To enrich for mouse zalpha11 5′ RACE product, a nested 5′ RACEreaction was performed using PCR reaction conditions as described abovefor the initial 5′RACE, except using nested primers ZC24,431 (SEQ IDNO:75) and ZC9,719 (SEQ ID NO:76), and one μl of a 1/20 dilution of theinitial 5′ RACE reaction (above) as a template. The products werepurified by gel electrophoresis, the DNA was eluted using the Qiaex IIAgarose Gel Extraction Kit (Qiagen) and subcloned using the TOPO TACloning Kit (Invitrogen). Positive clones were identified by colony PCRusing 10 pmol each of ZC24,431 (SEQ ID NO:75) and ZC24,511 (SEQ IDNO:77). The PCR reaction conditions were as follows: 94° C., 2 min.; 35cycles of 94° C. for 20 sec., 64° C. for 20 sec., and 72° C. for 30sec.; followed by one cycle at 72° C. for 5 min. Two subclones from eachof the nested 5′RACE reactions were sequenced. All the clones containedsome zalpha11 sequence but none were complete. A compiled sequence wasgenerated from the incomplete 5′RACE clones and the 3′ exon sequence(SEQ ID NO:70) representing a preliminary partial sequence of the mousezalpha11 polynucleotide and corresponding polypeptide. The preliminarysequence of the partial mouse zalpha11 cDNA is show in SEQ ID NO:78(5′end) and SEQ ID NO:80 (3′end); there was approximately 330nucleotides of yet unknown sequence between SEQ ID NO:78 (5′end) and SEQID NO:80 (3′end) to comprise the entire mouse zalpha11 cDNA (see below).The corresponding amino acid sequences for SEQ ID NO:78 and SEQ ID NO:80are shown in SEQ ID NO:79 (N-terminus) and SEQ ID NO:81 (C-terminus)respectively.

[0326] D. Full Length PCR

[0327] Primers were designed from the mouse upstream UTR of theinitiation Met and downstream of the termination codon for full lengthPCR. 20 pmol each of primers ZC24,616 (SEQ ID NO:82) and ZC24,615 (SEQID NO:83) were used in PCR reactions using a mouse dendritic cellmarathon cDNA or a neonatal mouse skin in-house cDNA library as atemplate. PCR reaction conditions were as follows: 94° C., 1 min.; 30cycles of 94° C. for 20 sec., and 66° C. for 2 min.; followed by onecycle at 72° C. for 5 min. The PCR products were purified by gelelectrophoresis, and the cDNA was eluted using the Qiaquick GelExtraction Kit and subcloned using the TA Cloning Kit (Invitrogen). 2subclones from each PCR reaction were sequenced. Sequencing primers usedwere: ZC694 (SEQ ID NO:87), ZC3,424 (SEQ ID NO:86), ZC24,431 (SEQ IDNO:75), ZC24,511 (SEQ ID NO:77), ZC24,806 (SEQ ID NO:90), and ZC24,807(SEQ ID NO:91). The sequence of the full length mouse zalpha11 cDNA isshow in SEQ ID NO:84. The corresponding amino acid sequence is shown inSEQ ID NO:85.

[0328] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 91 <210> SEQ ID NO 1<211> LENGTH: 2887 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (69)...(1682) <400>SEQUENCE: 1 gaagcagcag gtaccccctc cacatcccta gggctctgtg atgtaggcagaggcccgtgg 60 gagtcagc atg ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg ctgctg ctc 110 Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu 1 510 cag gga ggc tgg ggc tgc ccc gac ctc gtc tgc tac acc gat tac ctc 158Gln Gly Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu 15 20 2530 cag acg gtc atc tgc atc ctg gaa atg tgg aac ctc cac ccc agc acg 206Gln Thr Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr 35 40 45ctc acc ctt acc tgg caa gac cag tat gaa gag ctg aag gac gag gcc 254 LeuThr Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala 50 55 60 acctcc tgc agc ctc cac agg tcg gcc cac aat gcc acg cat gcc acc 302 Thr SerCys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr 65 70 75 tac acctgc cac atg gat gta ttc cac ttc atg gcc gac gac att ttc 350 Tyr Thr CysHis Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe 80 85 90 agt gtc aacatc aca gac cag tct ggc aac tac tcc cag gag tgt ggc 398 Ser Val Asn IleThr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly 95 100 105 110 agc tttctc ctg gct gag agc atc aag ccg gct ccc cct ttc aac gtg 446 Ser Phe LeuLeu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val 115 120 125 act gtgacc ttc tca gga cag tat aat atc tcc tgg cgc tca gat tac 494 Thr Val ThrPhe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr 130 135 140 gaa gaccct gcc ttc tac atg ctg aag ggc aag ctt cag tat gag ctg 542 Glu Asp ProAla Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu 145 150 155 cag tacagg aac cgg gga gac ccc tgg gct gtg agt ccg agg aga aag 590 Gln Tyr ArgAsn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys 160 165 170 ctg atctca gtg gac tca aga agt gtc tcc ctc ctc ccc ctg gag ttc 638 Leu Ile SerVal Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe 175 180 185 190 cgcaaa gac tcg agc tat gag ctg cag gtg cgg gca ggg ccc atg cct 686 Arg LysAsp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro 195 200 205 ggctcc tcc tac cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc 734 Gly SerSer Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile 210 215 220 tttcag acc cag tca gag gag tta aag gaa ggc tgg aac cct cac ctg 782 Phe GlnThr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu 225 230 235 ctgctt ctc ctc ctg ctt gtc ata gtc ttc att cct gcc ttc tgg agc 830 Leu LeuLeu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser 240 245 250 ctgaag acc cat cca ttg tgg agg cta tgg aag aag ata tgg gcc gtc 878 Leu LysThr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val 255 260 265 270ccc agc cct gag cgg ttc ttc atg ccc ctg tac aag ggc tgc agc gga 926 ProSer Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly 275 280 285gac ttc aag aaa tgg gtg ggt gca ccc ttc act ggc tcc agc ctg gag 974 AspPhe Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu 290 295 300ctg gga ccc tgg agc cca gag gtg ccc tcc acc ctg gag gtg tac agc 1022 LeuGly Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser 305 310 315tgc cac cca cca cgg agc ccg gcc aag agg ctg cag ctc acg gag cta 1070 CysHis Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu Leu 320 325 330caa gaa cca gca gag ctg gtg gag tct gac ggt gtg ccc aag ccc agc 1118 GlnGlu Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser 335 340 345350 ttc tgg ccg aca gcc cag aac tcg ggg ggc tca gct tac agt gag gag 1166Phe Trp Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu 355 360365 agg gat cgg cca tac ggc ctg gtg tcc att gac aca gtg act gtg cta 1214Arg Asp Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu 370 375380 gat gca gag ggg cca tgc acc tgg ccc tgc agc tgt gag gat gac ggc 1262Asp Ala Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly 385 390395 tac cca gcc ctg gac ctg gat gct ggc ctg gag ccc agc cca ggc cta 1310Tyr Pro Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu 400 405410 gag gac cca ctc ttg gat gca ggg acc aca gtc ctg tcc tgt ggc tgt 1358Glu Asp Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys 415 420425 430 gtc tca gct ggc agc cct ggg cta gga ggg ccc ctg gga agc ctc ctg1406 Val Ser Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser Leu Leu 435440 445 gac aga cta aag cca ccc ctt gca gat ggg gag gac tgg gct ggg gga1454 Asp Arg Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp Trp Ala Gly Gly 450455 460 ctg ccc tgg ggt ggc cgg tca cct gga ggg gtc tca gag agt gag gcg1502 Leu Pro Trp Gly Gly Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala 465470 475 ggc tca ccc ctg gcc ggc ctg gat atg gac acg ttt gac agt ggc ttt1550 Gly Ser Pro Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe 480485 490 gtg ggc tct gac tgc agc agc cct gtg gag tgt gac ttc acc agc ccc1598 Val Gly Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro 495500 505 510 ggg gac gaa gga ccc ccc cgg agc tac ctc cgc cag tgg gtg gtcatt 1646 Gly Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile515 520 525 cct ccg cca ctt tcg agc cct gga ccc cag gcc agc taatgaggct1692 Pro Pro Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser 530 535 gactggatgtccagagctgg ccaggccact gggccctgag ccagagacaa ggtcacctgg 1752 gctgtgatgtgaagacacct gcagcctttg gtctcctgga tgggcctttg agcctgatgt 1812 ttacagtgtctgtgtgtgtg tgtgcatatg tgtgtgtgtg catatgcatg tgtgtgtgtg 1872 tgtgtgtcttaggtgcgcag tggcatgtcc acgtgtgtgt gtgattgcac gtgcctgtgg 1932 gcctgggataatgcccatgg tactccatgc attcacctgc cctgtgcatg tctggactca 1992 cggagctcacccatgtgcac aagtgtgcac agtaaacgtg tttgtggtca acagatgaca 2052 acagccgtcctccctcctag ggtcttgtgt tgcaagttgg tccacagcat ctccggggct 2112 ttgtgggatcagggcattgc ctgtgactga ggcggagccc agccctccag cgtctgcctc 2172 caggagctgcaagaagtcca tattgttcct tatcacctgc caacaggaag cgaaagggga 2232 tggagtgagcccatggtgac ctcgggaatg gcaatttttt gggcggcccc tggacgaagg 2292 tctgaatcccgactctgata ccttctggct gtgctacctg agccaagtcg cctcccctct 2352 ctgggctagagtttccttat ccagacagtg gggaaggcat gacacacctg ggggaaattg 2412 gcgatgtcacccgtgtacgg tacgcagccc agagcagacc ctcaataaac gtcagcttcc 2472 ttccttctgcggccagagcc gaggcgggcg ggggtgagaa catcaatcgt cagcgacagc 2532 ctgggcacccgcggggccgt cccgcctgca gagggccact cgggggggtt tccaggctta 2592 aaatcagtccgtttcgtctc ttggaaacag ctccccacca accaagattt ctttttctaa 2652 cttctgctactaagttttta aaaattccct ttatgcaccc aagagatatt tattaaacac 2712 caattacgtagcaggccatg gctcatggga cccacccccc gtggcactca tggagggggc 2772 tgcaggttggaactatgcag tgtgctccgg ccacacatcc tgctgggccc cctaccctgc 2832 cccaattcaatcctgccaat aaatcctgtc ttatttgttc atcctggaga attga 2887 <210> SEQ ID NO 2<211> LENGTH: 538 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 2 Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu GlnGly 1 5 10 15 Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr LeuGln Thr 20 25 30 Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser ThrLeu Thr 35 40 45 Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu AlaThr Ser 50 55 60 Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala ThrTyr Thr 65 70 75 80 Cys His Met Asp Val Phe His Phe Met Ala Asp Asp IlePhe Ser Val 85 90 95 Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu CysGly Ser Phe 100 105 110 Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro PheAsn Val Thr Val 115 120 125 Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp ArgSer Asp Tyr Glu Asp 130 135 140 Pro Ala Phe Tyr Met Leu Lys Gly Lys LeuGln Tyr Glu Leu Gln Tyr 145 150 155 160 Arg Asn Arg Gly Asp Pro Trp AlaVal Ser Pro Arg Arg Lys Leu Ile 165 170 175 Ser Val Asp Ser Arg Ser ValSer Leu Leu Pro Leu Glu Phe Arg Lys 180 185 190 Asp Ser Ser Tyr Glu LeuGln Val Arg Ala Gly Pro Met Pro Gly Ser 195 200 205 Ser Tyr Gln Gly ThrTrp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220 Thr Gln Ser GluGlu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu 225 230 235 240 Leu LeuLeu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys 245 250 255 ThrHis Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser 260 265 270Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe 275 280285 Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly 290295 300 Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser Cys His305 310 315 320 Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu Thr Glu LeuGln Glu 325 330 335 Pro Ala Glu Leu Val Glu Ser Asp Gly Val Pro Lys ProSer Phe Trp 340 345 350 Pro Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr SerGlu Glu Arg Asp 355 360 365 Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr ValThr Val Leu Asp Ala 370 375 380 Glu Gly Pro Cys Thr Trp Pro Cys Ser CysGlu Asp Asp Gly Tyr Pro 385 390 395 400 Ala Leu Asp Leu Asp Ala Gly LeuGlu Pro Ser Pro Gly Leu Glu Asp 405 410 415 Pro Leu Leu Asp Ala Gly ThrThr Val Leu Ser Cys Gly Cys Val Ser 420 425 430 Ala Gly Ser Pro Gly LeuGly Gly Pro Leu Gly Ser Leu Leu Asp Arg 435 440 445 Leu Lys Pro Pro LeuAla Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro 450 455 460 Trp Gly Gly ArgSer Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser 465 470 475 480 Pro LeuAla Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly 485 490 495 SerAsp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp 500 505 510Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro 515 520525 Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser 530 535 <210> SEQ ID NO 3<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: consensus amino acid motif <220>FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(5) <223> OTHERINFORMATION: Xaa = Any Amino Acid <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(5) <223> OTHER INFORMATION: Xaa = Any Amino Acid<400> SEQUENCE: 3 Trp Ser Xaa Trp Ser 1 5 <210> SEQ ID NO 4 <211>LENGTH: 1614 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: degenerate nucleotide sequence ofzalpha11 <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)...(1614) <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1614) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 4 atgccnmgng gntgggcngcnccnytnytn ytnytnytny tncarggngg ntggggntgy 60 ccngayytng tntgytayacngaytayytn caracngtna thtgyathyt ngaratgtgg 120 aayytncayc cnwsnacnytnacnytnacn tggcargayc artaygarga rytnaargay 180 gargcnacnw sntgywsnytncaymgnwsn gcncayaayg cnacncaygc nacntayacn 240 tgycayatgg aygtnttycayttyatggcn gaygayatht tywsngtnaa yathacngay 300 carwsnggna aytaywsncargartgyggn wsnttyytny tngcngarws nathaarccn 360 gcnccnccnt tyaaygtnacngtnacntty wsnggncart ayaayathws ntggmgnwsn 420 gaytaygarg ayccngcnttytayatgytn aarggnaary tncartayga rytncartay 480 mgnaaymgng gngayccntgggcngtnwsn ccnmgnmgna arytnathws ngtngaywsn 540 mgnwsngtnw snytnytnccnytngartty mgnaargayw snwsntayga rytncargtn 600 mgngcnggnc cnatgccnggnwsnwsntay carggnacnt ggwsngartg gwsngayccn 660 gtnathttyc aracncarwsngargarytn aargarggnt ggaayccnca yytnytnytn 720 ytnytnytny tngtnathgtnttyathccn gcnttytggw snytnaarac ncayccnytn 780 tggmgnytnt ggaaraarathtgggcngtn ccnwsnccng armgnttytt yatgccnytn 840 tayaarggnt gywsnggngayttyaaraar tgggtnggng cnccnttyac nggnwsnwsn 900 ytngarytng gnccntggwsnccngargtn ccnwsnacny tngargtnta ywsntgycay 960 ccnccnmgnw snccngcnaarmgnytncar ytnacngary tncargarcc ngcngarytn 1020 gtngarwsng ayggngtnccnaarccnwsn ttytggccna cngcncaraa ywsnggnggn 1080 wsngcntayw sngargarmgngaymgnccn tayggnytng tnwsnathga yacngtnacn 1140 gtnytngayg cngarggnccntgyacntgg ccntgywsnt gygargayga yggntayccn 1200 gcnytngayy tngaygcnggnytngarccn wsnccnggny tngargaycc nytnytngay 1260 gcnggnacna cngtnytnwsntgyggntgy gtnwsngcng gnwsnccngg nytnggnggn 1320 ccnytnggnw snytnytngaymgnytnaar ccnccnytng cngayggnga rgaytgggcn 1380 ggnggnytnc cntggggnggnmgnwsnccn ggnggngtnw sngarwsnga rgcnggnwsn 1440 ccnytngcng gnytngayatggayacntty gaywsnggnt tygtnggnws ngaytgywsn 1500 wsnccngtng artgygayttyacnwsnccn ggngaygarg gnccnccnmg nwsntayytn 1560 mgncartggg tngtnathccnccnccnytn wsnwsnccng gnccncargc nwsn 1614 <210> SEQ ID NO 5 <211>LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC447 <400>SEQUENCE: 5 taacaatttc acacagg 17 <210> SEQ ID NO 6 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Oligonucleotide primer ZC976 <400> SEQUENCE: 6cgttgtaaaa cgacggcc 18 <210> SEQ ID NO 7 <211> LENGTH: 21 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC19345 <400> SEQUENCE: 7 gaccagtctggcaactactc c 21 <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19346 <400> SEQUENCE: 8 gctctcagcc aggagaaagc20 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19349 <400> SEQUENCE: 9 ggttggtggg gagctgtttc20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19350 <400> SEQUENCE: 10 gggtgagaac atcaatcgtc20 <210> SEQ ID NO 11 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19458 <400> SEQUENCE: 11 catatcttct tccatagcctc 21 <210> SEQ ID NO 12 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19459 <400> SEQUENCE: 12 ctcctcctgc ttgtcatagtc 21 <210> SEQ ID NO 13 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19460 <400> SEQUENCE: 13 gtaaacgtgt ttgtggtcaac 21 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19461 <400> SEQUENCE: 14 tgccctgatc ccacaaagcc20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19572 <400> SEQUENCE: 15 gtcctgtggc tgtgtctcag20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19573 <400> SEQUENCE: 16 cagtcagagc ccacaaagcc20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19657 <400> SEQUENCE: 17 ctgagacaca gccacaggac20 <210> SEQ ID NO 18 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19181 <400> SEQUENCE: 18 tccacatccc tagggctctgtgat 24 <210> SEQ ID NO 19 <211> LENGTH: 25 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19182 <400> SEQUENCE: 19 gaggttccac atttccaggatgcag 25 <210> SEQ ID NO 20 <211> LENGTH: 24 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19907 <400> SEQUENCE: 20 atggatgtat tccacttcatggcc 24 <210> SEQ ID NO 21 <211> LENGTH: 24 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19908 <400> SEQUENCE: 21 actgtcaaac gtgtccatatccag 24 <210> SEQ ID NO 22 <211> LENGTH: 18 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19954 <400> SEQUENCE: 22 actgggctgg gggactgc 18<210> SEQ ID NO 23 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19955 <400> SEQUENCE: 23 ccccggggct ggtgaagt 18<210> SEQ ID NO 24 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC17212 <400> SEQUENCE: 24 ggggaattcg aagccatgccctcttgggcc ctc 33 <210> SEQ ID NO 25 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC19914 <400> SEQUENCE: 25caatggatgg gtctttagca gcagtaggcc 30 <210> SEQ ID NO 26 <211> LENGTH: 30<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Oligonucleotide primer ZC19913 <400> SEQUENCE: 26ggcctactgc tgctaaagac ccatccattg 30 <210> SEQ ID NO 27 <211> LENGTH: 33<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Oligonucleotide primer ZC20097 <400> SEQUENCE: 27acatctagat tagctggcct ggggtccagg cgt 33 <210> SEQ ID NO 28 <211> LENGTH:21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer ZC12700 <400> SEQUENCE:28 ggaggtctat ataagcagag c 21 <210> SEQ ID NO 29 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC5020 <400> SEQUENCE: 29 cactggagtggcaacttcca g 21 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC6675 <400> SEQUENCE: 30 gtggatgccgaacccagtcc 20 <210> SEQ ID NO 31 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC7727 <400> SEQUENCE: 31 tgttcacagc tacctgggct c21 <210> SEQ ID NO 32 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC8290 <400> SEQUENCE: 32 ccaccgagac tgcttggatcaccttg 26 <210> SEQ ID NO 33 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC6622 <400> SEQUENCE: 33 ctgggctgga aactggcaca c21 <210> SEQ ID NO 34 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC7736 <400> SEQUENCE: 34 cactgtcaga aatggagc 18<210> SEQ ID NO 35 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC9273 <400> SEQUENCE: 35 ggtccctccc cgggcaccgagaga 24 <210> SEQ ID NO 36 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19905 <400> SEQUENCE: 36 acaggatccg tcagcatgccgcgtggctgg gccgcc 36 <210> SEQ ID NO 37 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC19906 <400> SEQUENCE: 37acagaattct tagctggcct ggggtccagg cgt 33 <210> SEQ ID NO 38 <211> LENGTH:22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer ZC20114 <400> SEQUENCE:38 cctgccttct acatgctgaa gg 22 <210> SEQ ID NO 39 <211> LENGTH: 18 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC19954 <400> SEQUENCE: 39actgggctgg gggactgc 18 <210> SEQ ID NO 40 <211> LENGTH: 22 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC20116 <400> SEQUENCE: 40agcacagtca ctgtgtcaat gg 22 <210> SEQ ID NO 41 <211> LENGTH: 6 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Glu-Glu (CEE) tag amino acid sequence <400> SEQUENCE: 41Glu Tyr Met Pro Met lu 1 5 <210> SEQ ID NO 42 <211> LENGTH: 36 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide promer ZC19931 <400> SEQUENCE: 42ggttggtacc gcaagatgcc gcgtggctgg gccgcc 36 <210> SEQ ID NO 43 <211>LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC19932 <400>SEQUENCE: 43 cggaggatcc gtgagggttc cagccttcc 29 <210> SEQ ID NO 44 <211>LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer spanning vectorflanking region and the 5′ end of the zalpha11 <400> SEQUENCE: 44tccactttgc ctttctctcc acaggtgtcc agggaattca tcgataatgc cgcgtggctg 60ggccgc 66 <210> SEQ ID NO 45 <211> LENGTH: 699 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <400> SEQUENCE: 45 gagcccagat cttcagacaaaactcacaca tgcccaccgt gcccagcacc tgaagccgag 60 ggggcaccgt cagtcttcctcttcccccca aaacccaagg acaccctcat gatctcccgg 120 acccctgagg tcacatgcgtggtggtggac gtgagccacg aagaccctga ggtcaagttc 180 aactggtacg tggacggcgtggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240 tacaacagca cgtaccgtgtggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300 ggcaaggagt acaagtgcaaggtctccaac aaagccctcc catcctccat cgagaaaacc 360 atctccaaag ccaaagggcagccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420 gatgagctga ccaagaaccaggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480 gacatcgccg tggagtgggagagcaatggg cagccggaga acaactacaa gaccacgcct 540 cccgtgctgg actccgacggctccttcttc ctctacagca agctcaccgt ggacaagagc 600 aggtggcagc aggggaacgtcttctcatgc tccgtgatgc atgaggctct gcacaaccac 660 tacacgcaga agagcctctccctgtctccg ggtaaataa 699 <210> SEQ ID NO 46 <211> LENGTH: 62 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: First Oligonucleotide primer spanning 3′ end of thezalpha11 extracellular domain and the 5′ end of Fc4 <400> SEQUENCE: 46gcacggtggg catgtgtgag ttttgtctga agatctgggc tcgtgagggt tccagccttc 60 ct62 <210> SEQ ID NO 47 <211> LENGTH: 61 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: SecondOligonucleotide primer spanning 3′ end of the zalpha11 extracellulardomain and the 5′ end of Fc4 <400> SEQUENCE: 47 agacccagtc agaggagttaaaggaaggct ggaaccctca cgagcccaga tcttcagaca 60 a 61 <210> SEQ ID NO 48<211> LENGTH: 67 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer spanningthe 3′ end of Fc4 and the vector flanking region <400> SEQUENCE: 48gtgggcctct ggggtgggta caaccccaga gctgttttaa tctagattat ttacccggag 60acaggga 67 <210> SEQ ID NO 49 <211> LENGTH: 8 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:FLAG tag amino acid sequence <400> SEQUENCE: 49 Asp Tyr Lys Asp Asp AspAsp Lys 1 5 <210> SEQ ID NO 50 <211> LENGTH: 1821 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Polynucleotide encoding MBP-zalpha11 soluble receptor fusion <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1821) <400>SEQUENCE: 50 atg aaa atc gaa gaa ggt aaa ctg gta atc tgg att aac ggc gataaa 48 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 15 10 15 ggc tat aac ggt ctc gct gaa gtc ggt aag aaa ttc gag aaa gat acc96 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 2530 gga att aaa gtc acc gtt gag cat ccg gat aaa ctg gaa gag aaa ttc 144Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45cca cag gtt gcg gca act ggc gat ggc cct gac att atc ttc tgg gca 192 ProGln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 cacgac cgc ttt ggt ggc tac gct caa tct ggc ctg ttg gct gaa atc 240 His AspArg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 accccg gac aaa gcg ttc cag gac aag ctg tat ccg ttt acc tgg gat 288 Thr ProAsp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 gcc gtacgt tac aac ggc aag ctg att gct tac ccg atc gct gtt gaa 336 Ala Val ArgTyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 gcg ttatcg ctg att tat aac aaa gat ctg ctg ccg aac ccg cca aaa 384 Ala Leu SerLeu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 acc tgggaa gag atc ccg gcg ctg gat aaa gaa ctg aaa gcg aaa ggt 432 Thr Trp GluGlu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 aag agcgcg ctg atg ttc aac ctg caa gaa ccg tac ttc acc tgg ccg 480 Lys Ser AlaLeu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 ctgatt gct gct gac ggg ggt tat gcg ttc aag tat gaa aac ggc aag 528 Leu IleAla Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 tacgac att aaa gac gtg ggc gtg gat aac gct ggc gcg aaa gcg ggt 576 Tyr AspIle Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 ctgacc ttc ctg gtt gac ctg att aaa aac aaa cac atg aat gca gac 624 Leu ThrPhe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 accgat tac tcc atc gca gaa gct gcc ttt aat aaa ggc gaa aca gcg 672 Thr AspTyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 atgacc atc aac ggc ccg tgg gca tgg tcc aac atc gac acc agc aaa 720 Met ThrIle Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240gtg aat tat ggt gta acg gta ctg ccg acc ttc aag ggt caa cca tcc 768 ValAsn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255aaa ccg ttc gtt ggc gtg ctg agc gca ggt att aac gcc gcc agt ccg 816 LysPro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270aac aaa gag ctg gca aaa gag ttc ctc gaa aac tat ctg ctg act gat 864 AsnLys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285gaa ggt ctg gaa gcg gtt aat aaa gac aaa ccg ctg ggt gcc gta gcg 912 GluGly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300ctg aag tct tac gag gaa gag ttg gcg aaa gat cca cgt att gcc gcc 960 LeuLys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315320 acc atg gaa aac gcc cag aaa ggt gaa atc atg ccg aac atc ccg cag 1008Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330335 atg tcc gct ttc tgg tat gcc gtg cgt act gcg gtg atc aac gcc gcc 1056Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345350 agc ggt cgt cag act gtc gat gaa gcc ctg aaa gac gcg cag act aat 1104Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360365 tcg agc tcc cac cat cac cat cac cac gcg aat tcg gta ccg ctg gtt 1152Ser Ser Ser His His His His His His Ala Asn Ser Val Pro Leu Val 370 375380 ccg cgt gga tcc tgc ccc gac ctc gtc tgc tac acc gat tac ctc cag 1200Pro Arg Gly Ser Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln 385 390395 400 acg gtc atc tgc atc ctg gaa atg tgg aac ctc cac ccc agc acg ctc1248 Thr Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu 405410 415 acc ctt acc tgg caa gac cag tat gaa gag ctg aag gac gag gcc acc1296 Thr Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr 420425 430 tcc tgc agc ctc cac agg tcg gcc cac aat gcc acg cat gcc acc tac1344 Ser Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr 435440 445 acc tgc cac atg gat gta ttc cac ttc atg gcc gac gac att ttc agt1392 Thr Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser 450455 460 gtc aac atc aca gac cag tct ggc aac tac tcc cag gag tgt ggc agc1440 Val Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser 465470 475 480 ttt ctc ctg gct gag agc atc aag ccg gct ccc cct ttc aac gtgact 1488 Phe Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr485 490 495 gtg acc ttc tca gga cag tat aat atc tcc tgg cgc tca gat tacgaa 1536 Val Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu500 505 510 gac cct gcc ttc tac atg ctg aag ggc aag ctt cag tat gag ctgcag 1584 Asp Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln515 520 525 tac agg aac cgg gga gac ccc tgg gct gtg agt ccg agg aga aagctg 1632 Tyr Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu530 535 540 atc tca gtg gac tca aga agt gtc tcc ctc ctc ccc ctg gag ttccgc 1680 Ile Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg545 550 555 560 aaa gac tcg agc tat gag ctg cag gtg cgg gca ggg ccc atgcct ggc 1728 Lys Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met ProGly 565 570 575 tcc tcc tac cag ggg acc tgg agt gaa tgg agt gac ccg gtcatc ttt 1776 Ser Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val IlePhe 580 585 590 cag acc cag tca gag gag tta aag gaa ggc tgg aac cct cactag 1821 Gln Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His 595 600605 <210> SEQ ID NO 51 <211> LENGTH: 606 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: : <400>SEQUENCE: 51 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly AspLys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu LysAsp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu GluLys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile PheTrp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu AlaGlu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro PheThr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro IleAla Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu ProAsn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys GluLeu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln GluPro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly TyrAla Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val GlyVal Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val AspLeu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser IleAla Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile AsnGly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val AsnTyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 LysPro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn IlePro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val IleAsn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys AspAla Gln Thr Asn 355 360 365 Ser Ser Ser His His His His His His Ala AsnSer Val Pro Leu Val 370 375 380 Pro Arg Gly Ser Cys Pro Asp Leu Val CysTyr Thr Asp Tyr Leu Gln 385 390 395 400 Thr Val Ile Cys Ile Leu Glu MetTrp Asn Leu His Pro Ser Thr Leu 405 410 415 Thr Leu Thr Trp Gln Asp GlnTyr Glu Glu Leu Lys Asp Glu Ala Thr 420 425 430 Ser Cys Ser Leu His ArgSer Ala His Asn Ala Thr His Ala Thr Tyr 435 440 445 Thr Cys His Met AspVal Phe His Phe Met Ala Asp Asp Ile Phe Ser 450 455 460 Val Asn Ile ThrAsp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser 465 470 475 480 Phe LeuLeu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr 485 490 495 ValThr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu 500 505 510Asp Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln 515 520525 Tyr Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu 530535 540 Ile Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg545 550 555 560 Lys Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro MetPro Gly 565 570 575 Ser Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp ProVal Ile Phe 580 585 590 Gln Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp AsnPro His 595 600 605 <210> SEQ ID NO 52 <211> LENGTH: 657 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 52 tgccccgacc tcgtctgctacaccgattac ctccagacgg tcatctgcat cctggaaatg 60 tggaacctcc accccagcacgctcaccctt acctggcaag accagtatga agagctgaag 120 gacgaggcca cctcctgcagcctccacagg tcggcccaca atgccacgca tgccacctac 180 acctgccaca tggatgtattccacttcatg gccgacgaca ttttcagtgt caacatcaca 240 gaccagtctg gcaactactcccaggagtgt ggcagctttc tcctggctga gagcatcaag 300 ccggctcccc ctttcaacgtgactgtgacc ttctcaggac agtataatat ctcctggcgc 360 tcagattacg aagaccctgccttctacatg ctgaagggca agcttcagta tgagctgcag 420 tacaggaacc ggggagacccctgggctgtg agtccgagga gaaagctgat ctcagtggac 480 tcaagaagtg tctccctcctccccctggag ttccgcaaag actcgagcta tgagctgcag 540 gtgcgggcag ggcccatgcctggctcctcc taccagggga cctggagtga atggagtgac 600 ccggtcatct ttcagacccagtcagaggag ttaaaggaag gctggaaccc tcactag 657 <210> SEQ ID NO 53 <211>LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC20187 <400>SEQUENCE: 53 tcaccacgcg aattcggtac cgctggttcc gcgtggatcc tgccccgacctcgtctgcta 60 caccg 65 <210> SEQ ID NO 54 <211> LENGTH: 68 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC20185 <400> SEQUENCE: 54tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca ctagtgaggg ttccagcctt 60cctttaac 68 <210> SEQ ID NO 55 <211> LENGTH: 40 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19372 <400> SEQUENCE: 55 tgtcgatgaa gccctgaaagacgcgcagac taattcgagc 40 <210> SEQ ID NO 56 <211> LENGTH: 60 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC19351 <400> SEQUENCE: 56acgcgcagac taattcgagc tcccaccatc accatcacca cgcgaattcg gtaccgctgg 60<210> SEQ ID NO 57 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC19352 <400> SEQUENCE: 57 actcactata gggcgaattgcccgggggat ccacgcggaa ccagcggtac cgaattcgcg 60 <210> SEQ ID NO 58 <211>LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC19371 <400>SEQUENCE: 58 acggccagtg aattgtaata cgactcacta tagggcgaat tg 42 <210> SEQID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide primerZC22277 <400> SEQUENCE: 59 ccaggagtgt ggcagctttc 20 <210> SEQ ID NO 60<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC22276<400> SEQUENCE: 60 gcttgccctt cagcatgtag a 21 <210> SEQ ID NO 61 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: zalpha11 TaqMan probe, ZG31 <400>SEQUENCE: 61 cggctccccc tttcaacgtg act 23 <210> SEQ ID NO 62 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC23684 <400>SEQUENCE: 62 tcacccttac ctggcaagac 20 <210> SEQ ID NO 63 <211> LENGTH:41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer ZC23656 <400> SEQUENCE:63 taatacgact cactataggg agggggagac acttcttgag t 41 <210> SEQ ID NO 64<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC23685<400> SEQUENCE: 64 aggtctgaat cccgactctg 20 <210> SEQ ID NO 65 <211>LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC23657 <400>SEQUENCE: 65 taatacgact cactataggg aggacgtaat tggtgtttaa t 41 <210> SEQID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer,rRNA forward primer <400> SEQUENCE: 66 cggctaccac atccaaggaa 20 <210>SEQ ID NO 67 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer,rRNA reverse primer <400> SEQUENCE: 67 gctggaatta ccgcggct 18 <210> SEQID NO 68 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: rRNA TaqMan probe <400>SEQUENCE: 68 tgctggcacc agacttgccc tc 22 <210> SEQ ID NO 69 <211>LENGTH: 1298 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (543)...(1262) <400> SEQUENCE: 69aggcctttca acacggcttt ttagtaattc attccatcta taaacattta tggtacacct 60actgtgtgcc aggtactgag gacacagttg tgatcagggc tagtgtagac acacaagcaa 120aactagagac atccggaagt gtcaggagac ggagtagagg ctgggccact tagacctcag 180gctctccctg cacacgtcct caagacctta ggacttagga acctggtccc agcacccagc 240tgttccttgg ctggggcact ggtaactagc gtggatatga gacagaggac agtcagtcct 300tactaaaggt gggaacacgg gctctgagaa cggacagtat tgggaaccca ctgggcaggg 360ggttcacaga cagacatcat ggcgcgctct ctctctctct ctctctcctg ttttcttgtt 420cttctgcttt ccccgtctct ggcttgtccc tgtactcccc cccccacccc catctttggc 480tctctctgtt cacacccgac cttgttgtcc ccagctcatg actgtgtgtt tctttctcat 540 agaaa tgg gtt aat acc cct ttc acg gcc tcc agc ata gag ttg gtg 587 Lys TrpVal Asn Thr Pro Phe Thr Ala Ser Ser Ile Glu Leu Val 1 5 10 15 cca cagagt tcc aca aca aca tca gcc tta cat ctg tca ttg tat cca 635 Pro Gln SerSer Thr Thr Thr Ser Ala Leu His Leu Ser Leu Tyr Pro 20 25 30 gcc aag gagaag aag ttc ccg ggg ctg ccg ggt ctg gaa gag caa ctg 683 Ala Lys Glu LysLys Phe Pro Gly Leu Pro Gly Leu Glu Glu Gln Leu 35 40 45 gag tgt gat ggaatg tct gag cct ggt cac tgg tgc ata atc ccc ttg 731 Glu Cys Asp Gly MetSer Glu Pro Gly His Trp Cys Ile Ile Pro Leu 50 55 60 gca gct ggc caa gcggtc tca gcc tac agt gag gag aga gac cgg cca 779 Ala Ala Gly Gln Ala ValSer Ala Tyr Ser Glu Glu Arg Asp Arg Pro 65 70 75 tat ggt ctg gtg tcc attgac aca gtg act gtg gga gat gca gag ggc 827 Tyr Gly Leu Val Ser Ile AspThr Val Thr Val Gly Asp Ala Glu Gly 80 85 90 95 ctg tgt gtc tgg ccc tgtagc tgt gag gat gat ggc tat cca gcc atg 875 Leu Cys Val Trp Pro Cys SerCys Glu Asp Asp Gly Tyr Pro Ala Met 100 105 110 aac ctg gat gct ggc cgagag tct ggc cct aat tca gag gat ctg ctc 923 Asn Leu Asp Ala Gly Arg GluSer Gly Pro Asn Ser Glu Asp Leu Leu 115 120 125 ttg gtc aca gac cct gctttt ctg tct tgc ggc tgt gtc tca ggt agt 971 Leu Val Thr Asp Pro Ala PheLeu Ser Cys Gly Cys Val Ser Gly Ser 130 135 140 ggt ctc agg ctt gga ggctcc cca ggc agc cta ctg gac agg ttg agg 1019 Gly Leu Arg Leu Gly Gly SerPro Gly Ser Leu Leu Asp Arg Leu Arg 145 150 155 ctg tca ttt gca aag gaaggg gac tgg aca gca gac cca acc tgg aga 1067 Leu Ser Phe Ala Lys Glu GlyAsp Trp Thr Ala Asp Pro Thr Trp Arg 160 165 170 175 act ggg tcc cca ggaggg ggc tct gag agt gaa gca ggt tcc ccc cct 1115 Thr Gly Ser Pro Gly GlyGly Ser Glu Ser Glu Ala Gly Ser Pro Pro 180 185 190 ggt ctg gac atg gacaca ttt gac agt ggc ttt gca ggt tca gac tgt 1163 Gly Leu Asp Met Asp ThrPhe Asp Ser Gly Phe Ala Gly Ser Asp Cys 195 200 205 ggc agc ccc gtg gagact gat gaa gga ccc cct cga agc tat ctc cgc 1211 Gly Ser Pro Val Glu ThrAsp Glu Gly Pro Pro Arg Ser Tyr Leu Arg 210 215 220 cag tgg gtg gtc aggacc cct cca cct gtg gac agt gga gcc cag agc 1259 Gln Trp Val Val Arg ThrPro Pro Pro Val Asp Ser Gly Ala Gln Ser 225 230 235 agc tagcatataataaccagcta tagtgagaag aggcct 1298 Ser 240 <210> SEQ ID NO 70 <211>LENGTH: 240 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:70 Lys Trp Val Asn Thr Pro Phe Thr Ala Ser Ser Ile Glu Leu Val Pro 1 510 15 Gln Ser Ser Thr Thr Thr Ser Ala Leu His Leu Ser Leu Tyr Pro Ala 2025 30 Lys Glu Lys Lys Phe Pro Gly Leu Pro Gly Leu Glu Glu Gln Leu Glu 3540 45 Cys Asp Gly Met Ser Glu Pro Gly His Trp Cys Ile Ile Pro Leu Ala 5055 60 Ala Gly Gln Ala Val Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro Tyr 6570 75 80 Gly Leu Val Ser Ile Asp Thr Val Thr Val Gly Asp Ala Glu Gly Leu85 90 95 Cys Val Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Met Asn100 105 110 Leu Asp Ala Gly Arg Glu Ser Gly Pro Asn Ser Glu Asp Leu LeuLeu 115 120 125 Val Thr Asp Pro Ala Phe Leu Ser Cys Gly Cys Val Ser GlySer Gly 130 135 140 Leu Arg Leu Gly Gly Ser Pro Gly Ser Leu Leu Asp ArgLeu Arg Leu 145 150 155 160 Ser Phe Ala Lys Glu Gly Asp Trp Thr Ala AspPro Thr Trp Arg Thr 165 170 175 Gly Ser Pro Gly Gly Gly Ser Glu Ser GluAla Gly Ser Pro Pro Gly 180 185 190 Leu Asp Met Asp Thr Phe Asp Ser GlyPhe Ala Gly Ser Asp Cys Gly 195 200 205 Ser Pro Val Glu Thr Asp Glu GlyPro Pro Arg Ser Tyr Leu Arg Gln 210 215 220 Trp Val Val Arg Thr Pro ProPro Val Asp Ser Gly Ala Gln Ser Ser 225 230 235 240 <210> SEQ ID NO 71<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC24432<400> SEQUENCE: 71 atgtctgagc ctggtcactg gtg 23 <210> SEQ ID NO 72 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC24433 <400>SEQUENCE: 72 tctgaacctg caaagccact gtc 23 <210> SEQ ID NO 73 <211>LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC9739 <400>SEQUENCE: 73 ccatcctaat acgactcact atagggc 27 <210> SEQ ID NO 74 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC24434 <400>SEQUENCE: 74 caccagtgac caggctcaga ca 22 <210> SEQ ID NO 75 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC24431 <400>SEQUENCE: 75 ccatcacact ccagttgctc ttc 23 <210> SEQ ID NO 76 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC9719 <400>SEQUENCE: 76 actcactata gggctcgagc ggc 23 <210> SEQ ID NO 77 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Oligonucleotide primer ZC24511 <400>SEQUENCE: 77 tccagcatag agttggtgcc aca 23 <210> SEQ ID NO 78 <211>LENGTH: 592 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (436)...(592) <400> SEQUENCE: 78cgcccgggca ggtctccgct ggtggccctg tgtttcagtc gcgcacagct gtctgcccac 60ttctcctgtg gtgtgcctca cggtcacttg cttgtctgac cgcaagtctg cccatccctg 120gggcagccaa ctggcctcag cccgtgcccc aggcgtgccc tgtctctgtc tggctgcccc 180agccctactg tcttcctctg tgtaggctct gcccagatgc ccggctggtc ctcagcctca 240ggactatctc agcagtgact cccctgattc tggacttgca cctgactgaa ctcctgccca 300cctcaaacct tcacctccca ccaccaccac tccgagtccc gctgtgactc ccacgcccag 360gagaccaccc aagtgcccca gcctaaagaa tggctttctg aggaagatcc tgaaggagta 420ggtctgggac acagc atg ccc cgg ggc cca gtg gct gcc tta ctc ctg ctg 471 MetPro Arg Gly Pro Val Ala Ala Leu Leu Leu Leu 1 5 10 att ctc cat gga gcttgg agc tgc ctg grc ctc act tgc tac act gac 519 Ile Leu His Gly Ala TrpSer Cys Leu Xaa Leu Thr Cys Tyr Thr Asp 15 20 25 tac ctc tgg acc atc acctgt gtc ctg gag aca cgg agc ccc aac ccc 567 Tyr Leu Trp Thr Ile Thr CysVal Leu Glu Thr Arg Ser Pro Asn Pro 30 35 40 agc ata ctc agt ctc acc tggcaa g 592 Ser Ile Leu Ser Leu Thr Trp Gln 45 50 <210> SEQ ID NO 79 <211>LENGTH: 52 <212> TYPE: PRT <213> ORGANISM: Mus musculus <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (1)...(51) <223> OTHERINFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 79 Met Pro Arg Gly ProVal Ala Ala Leu Leu Leu Leu Ile Leu His Gly 1 5 10 15 Ala Trp Ser CysLeu Xaa Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr 20 25 30 Ile Thr Cys ValLeu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu Ser 35 40 45 Leu Thr Trp Gln50 <210> SEQ ID NO 80 <211> LENGTH: 1229 <212> TYPE: DNA <213> ORGANISM:Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(3)...(1196) <400> SEQUENCE: 80 ga cgc tat gat atc tcc tgg gac tca gcttat gac gaa ccc tcc aac 47 Arg Tyr Asp Ile Ser Trp Asp Ser Ala Tyr AspGlu Pro Ser Asn 1 5 10 15 tac gtg ctg aga ggc aag cta caa tat gag ctgcag tat cgg aac ctc 95 Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu GlnTyr Arg Asn Leu 20 25 30 aga gac ccc tat gct gtg agg ccg gtg acc aag ctgatc tca gtg gac 143 Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu IleSer Val Asp 35 40 45 tca aga aac gtc tct ctt ctc cct gaa gag ttc cac aaagat tct agc 191 Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys AspSer Ser 50 55 60 tac cag ctg cag atg cgg gca gcg cct cag cca ggc act tcattc agg 239 Tyr Gln Leu Gln Met Arg Ala Ala Pro Gln Pro Gly Thr Ser PheArg 65 70 75 ggg acc tgg agt gag tgg agt gac ccc gtc atc ttt cgg acc caggct 287 Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Arg Thr Gln Ala80 85 90 95 ggg gag ccc gag gca ggc tgg gac cct cac atg ctg ctg ctc ctggct 335 Gly Glu Pro Glu Ala Gly Trp Asp Pro His Met Leu Leu Leu Leu Ala100 105 110 gtc ttg atc att gtc ctg gtt ttc atg ggt ctg aag atc cac ctgcct 383 Val Leu Ile Ile Val Leu Val Phe Met Gly Leu Lys Ile His Leu Pro115 120 125 tgg agg cta tgg aaa aag ata tgg gca cca gtg ccc acc cct gagagt 431 Trp Arg Leu Trp Lys Lys Ile Trp Ala Pro Val Pro Thr Pro Glu Ser130 135 140 ttc ttc cag ccc ctg tgc agg gag cac agc ggg aac ttc aag aaatgg 479 Phe Phe Gln Pro Leu Cys Arg Glu His Ser Gly Asn Phe Lys Lys Trp145 150 155 gtt aat acc cct ttc acg gcc tcc agc ata gag ttg gtg cca cagagt 527 Val Asn Thr Pro Phe Thr Ala Ser Ser Ile Glu Leu Val Pro Gln Ser160 165 170 175 tcc aca aca aca tca gcc tta cat ctg tca ttg tat cca gccaag gag 575 Ser Thr Thr Thr Ser Ala Leu His Leu Ser Leu Tyr Pro Ala LysGlu 180 185 190 aag aag ttc ccg ggg ctg ccg ggt ctg gaa gag caa ctg gagtgt gat 623 Lys Lys Phe Pro Gly Leu Pro Gly Leu Glu Glu Gln Leu Glu CysAsp 195 200 205 gga atg tct gag cct ggt cac tgg tgc ata atc ccc ttg gcagct ggc 671 Gly Met Ser Glu Pro Gly His Trp Cys Ile Ile Pro Leu Ala AlaGly 210 215 220 caa gcg gtc tca gcc tac agt gag gag aga gac cgg cca tatggt ctg 719 Gln Ala Val Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro Tyr GlyLeu 225 230 235 gtg tcc att gac aca gtg act gtg gga gat gca gag ggc ctgtgt gtc 767 Val Ser Ile Asp Thr Val Thr Val Gly Asp Ala Glu Gly Leu CysVal 240 245 250 255 tgg ccc tgt agc tgt gag gat gat ggc tat cca gcc atgaac ctg gat 815 Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Met AsnLeu Asp 260 265 270 gct ggc cga gag tct ggc cct aat tca gag gat ctg ctcttg gtc aca 863 Ala Gly Arg Glu Ser Gly Pro Asn Ser Glu Asp Leu Leu LeuVal Thr 275 280 285 gac cct gct ttt ctg tct tgc ggc tgt gtc tca ggt agtggt ctc agg 911 Asp Pro Ala Phe Leu Ser Cys Gly Cys Val Ser Gly Ser GlyLeu Arg 290 295 300 ctt gga ggc tcc cca ggc agc cta ctg gac agg ttg aggctg tca ttt 959 Leu Gly Gly Ser Pro Gly Ser Leu Leu Asp Arg Leu Arg LeuSer Phe 305 310 315 gca aag gaa ggg gac tgg aca gca gac cca acc tgg agaact ggg tcc 1007 Ala Lys Glu Gly Asp Trp Thr Ala Asp Pro Thr Trp Arg ThrGly Ser 320 325 330 335 cca gga ggg ggc tct gag agt gaa gca ggt tcc ccccct ggt ctg gac 1055 Pro Gly Gly Gly Ser Glu Ser Glu Ala Gly Ser Pro ProGly Leu Asp 340 345 350 atg gac aca ttt gac agt ggc ttt gca ggt tca gactgt ggc agc ccc 1103 Met Asp Thr Phe Asp Ser Gly Phe Ala Gly Ser Asp CysGly Ser Pro 355 360 365 gtg gag act gat gaa gga ccc cct cga agc tat ctccgc cag tgg gtg 1151 Val Glu Thr Asp Glu Gly Pro Pro Arg Ser Tyr Leu ArgGln Trp Val 370 375 380 gtc agg acc cct cca cct gtg gac agt gga gcc cagagc agc tag 1196 Val Arg Thr Pro Pro Pro Val Asp Ser Gly Ala Gln Ser Ser385 390 395 catataataa ccagctatag tgagaagagg cct 1229 <210> SEQ ID NO 81<211> LENGTH: 397 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 81 Arg Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu Pro Ser AsnTyr 1 5 10 15 Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg AsnLeu Arg 20 25 30 Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile Ser ValAsp Ser 35 40 45 Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys Asp SerSer Tyr 50 55 60 Gln Leu Gln Met Arg Ala Ala Pro Gln Pro Gly Thr Ser PheArg Gly 65 70 75 80 Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Arg ThrGln Ala Gly 85 90 95 Glu Pro Glu Ala Gly Trp Asp Pro His Met Leu Leu LeuLeu Ala Val 100 105 110 Leu Ile Ile Val Leu Val Phe Met Gly Leu Lys IleHis Leu Pro Trp 115 120 125 Arg Leu Trp Lys Lys Ile Trp Ala Pro Val ProThr Pro Glu Ser Phe 130 135 140 Phe Gln Pro Leu Cys Arg Glu His Ser GlyAsn Phe Lys Lys Trp Val 145 150 155 160 Asn Thr Pro Phe Thr Ala Ser SerIle Glu Leu Val Pro Gln Ser Ser 165 170 175 Thr Thr Thr Ser Ala Leu HisLeu Ser Leu Tyr Pro Ala Lys Glu Lys 180 185 190 Lys Phe Pro Gly Leu ProGly Leu Glu Glu Gln Leu Glu Cys Asp Gly 195 200 205 Met Ser Glu Pro GlyHis Trp Cys Ile Ile Pro Leu Ala Ala Gly Gln 210 215 220 Ala Val Ser AlaTyr Ser Glu Glu Arg Asp Arg Pro Tyr Gly Leu Val 225 230 235 240 Ser IleAsp Thr Val Thr Val Gly Asp Ala Glu Gly Leu Cys Val Trp 245 250 255 ProCys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Met Asn Leu Asp Ala 260 265 270Gly Arg Glu Ser Gly Pro Asn Ser Glu Asp Leu Leu Leu Val Thr Asp 275 280285 Pro Ala Phe Leu Ser Cys Gly Cys Val Ser Gly Ser Gly Leu Arg Leu 290295 300 Gly Gly Ser Pro Gly Ser Leu Leu Asp Arg Leu Arg Leu Ser Phe Ala305 310 315 320 Lys Glu Gly Asp Trp Thr Ala Asp Pro Thr Trp Arg Thr GlySer Pro 325 330 335 Gly Gly Gly Ser Glu Ser Glu Ala Gly Ser Pro Pro GlyLeu Asp Met 340 345 350 Asp Thr Phe Asp Ser Gly Phe Ala Gly Ser Asp CysGly Ser Pro Val 355 360 365 Glu Thr Asp Glu Gly Pro Pro Arg Ser Tyr LeuArg Gln Trp Val Val 370 375 380 Arg Thr Pro Pro Pro Val Asp Ser Gly AlaGln Ser Ser 385 390 395 <210> SEQ ID NO 82 <211> LENGTH: 23 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC24616 <400> SEQUENCE: 82ctgcccacct caaaccttca cct 23 <210> SEQ ID NO 83 <211> LENGTH: 24 .<212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Oligonucleotide primer ZC24615 <400> SEQUENCE: 83atgctagctg ctctgggctc cact 24 <210> SEQ ID NO 84 <211> LENGTH: 1735<212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (143)...(1729) <400> SEQUENCE: 84ctgcccacct caaaccttca cctcccacca ccaccactcc gagtcccgct gtgactccca 60cgcccaggag accacccaag tgccccagcc taaagaatgg ctttctgaga aagaccctga 120aggagtaggt ctgggacaca gc atg ccc cgg ggc cca gtg gct gcc tta ctc 172 MetPro Arg Gly Pro Val Ala Ala Leu Leu 1 5 10 ctg ctg att ctc cat gga gcttgg agc tgc ctg gac ctc act tgc tac 220 Leu Leu Ile Leu His Gly Ala TrpSer Cys Leu Asp Leu Thr Cys Tyr 15 20 25 act gac tac ctc tgg acc atc acctgt gtc ctg gag aca cgg agc ccc 268 Thr Asp Tyr Leu Trp Thr Ile Thr CysVal Leu Glu Thr Arg Ser Pro 30 35 40 aac ccc agc ata ctc agt ctc acc tggcaa gat gaa tat gag gaa ctt 316 Asn Pro Ser Ile Leu Ser Leu Thr Trp GlnAsp Glu Tyr Glu Glu Leu 45 50 55 cag gac caa gag acc ttc tgc agc cta cacagg tct ggc cac aac acc 364 Gln Asp Gln Glu Thr Phe Cys Ser Leu His ArgSer Gly His Asn Thr 60 65 70 aca cat ata tgg tac acg tgc cat atg cgc ttgtct caa ttc ctg tcc 412 Thr His Ile Trp Tyr Thr Cys His Met Arg Leu SerGln Phe Leu Ser 75 80 85 90 gat gaa gtt ttc att gtc aat gtg acg gac cagtct ggc aac aac tcc 460 Asp Glu Val Phe Ile Val Asn Val Thr Asp Gln SerGly Asn Asn Ser 95 100 105 caa gag tgt ggc agc ttt gtc ctg gct gag agcatc aaa cca gct ccc 508 Gln Glu Cys Gly Ser Phe Val Leu Ala Glu Ser IleLys Pro Ala Pro 110 115 120 ccc ttg aac gtg act gtg gcc ttc tca gga cgctat gat atc tcc tgg 556 Pro Leu Asn Val Thr Val Ala Phe Ser Gly Arg TyrAsp Ile Ser Trp 125 130 135 gac tca gct tat gac gaa ccc tcc aac tac gtgctg agg ggc aag cta 604 Asp Ser Ala Tyr Asp Glu Pro Ser Asn Tyr Val LeuArg Gly Lys Leu 140 145 150 caa tat gag ctg cag tat cgg aac ctc aga gacccc tat gct gtg agg 652 Gln Tyr Glu Leu Gln Tyr Arg Asn Leu Arg Asp ProTyr Ala Val Arg 155 160 165 170 ccg gtg acc aag ctg atc tca gtg gac tcaaga aac gtc tct ctt ctc 700 Pro Val Thr Lys Leu Ile Ser Val Asp Ser ArgAsn Val Ser Leu Leu 175 180 185 cct gaa gag ttc cac aaa gat tct agc taccag ctg cag gtg cgg gca 748 Pro Glu Glu Phe His Lys Asp Ser Ser Tyr GlnLeu Gln Val Arg Ala 190 195 200 gcg cct cag cca ggc act tca ttc agg gggacc tgg agt gag tgg agt 796 Ala Pro Gln Pro Gly Thr Ser Phe Arg Gly ThrTrp Ser Glu Trp Ser 205 210 215 gac ccc gtc atc ttt cag acc cag gct ggggag ccc gag gca ggc tgg 844 Asp Pro Val Ile Phe Gln Thr Gln Ala Gly GluPro Glu Ala Gly Trp 220 225 230 gac cct cac atg ctg ctg ctc ctg gct gtcttg atc att gtc ctg gtt 892 Asp Pro His Met Leu Leu Leu Leu Ala Val LeuIle Ile Val Leu Val 235 240 245 250 ttc atg ggt ctg aag atc cac ctg ccttgg agg cta tgg aaa aag ata 940 Phe Met Gly Leu Lys Ile His Leu Pro TrpArg Leu Trp Lys Lys Ile 255 260 265 tgg gca cca gtg ccc acc cct gag agtttc ttc cag ccc ctg tac agg 988 Trp Ala Pro Val Pro Thr Pro Glu Ser PhePhe Gln Pro Leu Tyr Arg 270 275 280 gag cac agc ggg aac ttc aag aaa tgggtt aat acc cct ttc acg gcc 1036 Glu His Ser Gly Asn Phe Lys Lys Trp ValAsn Thr Pro Phe Thr Ala 285 290 295 tcc agc ata gag ttg gtg cca cag agttcc aca aca aca tca gcc tta 1084 Ser Ser Ile Glu Leu Val Pro Gln Ser SerThr Thr Thr Ser Ala Leu 300 305 310 cat ctg tca ttg tat cca gcc aag gagaag aag ttc ccg ggg ctg ccg 1132 His Leu Ser Leu Tyr Pro Ala Lys Glu LysLys Phe Pro Gly Leu Pro 315 320 325 330 ggt ctg gaa gag caa ctg gag tgtgat gga atg tct gag cct ggt cac 1180 Gly Leu Glu Glu Gln Leu Glu Cys AspGly Met Ser Glu Pro Gly His 335 340 345 tgg tgc ata atc ccc ttg gca gctggc caa gcg gtc tca gcc tac agt 1228 Trp Cys Ile Ile Pro Leu Ala Ala GlyGln Ala Val Ser Ala Tyr Ser 350 355 360 gag gag aga gac cgg cca tat ggtctg gtg tcc att gac aca gtg act 1276 Glu Glu Arg Asp Arg Pro Tyr Gly LeuVal Ser Ile Asp Thr Val Thr 365 370 375 gtg gga gat gca gag ggc ctg tgtgtc tgg ccc tgt agc tgt gag gat 1324 Val Gly Asp Ala Glu Gly Leu Cys ValTrp Pro Cys Ser Cys Glu Asp 380 385 390 gat ggc tat cca gcc atg aac ctggat gct ggc cga gag tct ggc cct 1372 Asp Gly Tyr Pro Ala Met Asn Leu AspAla Gly Arg Glu Ser Gly Pro 395 400 405 410 aat tca gag gat ctg ctc ttggtc aca gac cct gct ttt ctg tct tgc 1420 Asn Ser Glu Asp Leu Leu Leu ValThr Asp Pro Ala Phe Leu Ser Cys 415 420 425 ggc tgt gtc tca ggt agt ggtctc agg ctt gga ggc tcc cca ggc agc 1468 Gly Cys Val Ser Gly Ser Gly LeuArg Leu Gly Gly Ser Pro Gly Ser 430 435 440 cta ctg gac agg ttg agg ctgtca ttt gca aag gaa ggg gac tgg aca 1516 Leu Leu Asp Arg Leu Arg Leu SerPhe Ala Lys Glu Gly Asp Trp Thr 445 450 455 gca gac cca acc tgg aga actggg tcc cca gga ggg ggc tct gag agt 1564 Ala Asp Pro Thr Trp Arg Thr GlySer Pro Gly Gly Gly Ser Glu Ser 460 465 470 gaa gca ggt tcc ccc cct ggtctg gac atg gac aca ttt gac agt ggc 1612 Glu Ala Gly Ser Pro Pro Gly LeuAsp Met Asp Thr Phe Asp Ser Gly 475 480 485 490 ttt gca ggt tca gac tgtggc agc ccc gtg gag act gat gaa gga ccc 1660 Phe Ala Gly Ser Asp Cys GlySer Pro Val Glu Thr Asp Glu Gly Pro 495 500 505 cct cga agc tat ctc cgccag tgg gtg gtc agg acc cct cca cct gtg 1708 Pro Arg Ser Tyr Leu Arg GlnTrp Val Val Arg Thr Pro Pro Pro Val 510 515 520 gac agt gga gcc cag agcagc tagcat 1735 Asp Ser Gly Ala Gln Ser Ser 525 <210> SEQ ID NO 85 <211>LENGTH: 529 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:85 Met Pro Arg Gly Pro Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly 1 510 15 Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr 2025 30 Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu Ser 3540 45 Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln Glu Thr Phe 5055 60 Cys Ser Leu His Arg Ser Gly His Asn Thr Thr His Ile Trp Tyr Thr 6570 75 80 Cys His Met Arg Leu Ser Gln Phe Leu Ser Asp Glu Val Phe Ile Val85 90 95 Asn Val Thr Asp Gln Ser Gly Asn Asn Ser Gln Glu Cys Gly Ser Phe100 105 110 Val Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Leu Asn Val ThrVal 115 120 125 Ala Phe Ser Gly Arg Tyr Asp Ile Ser Trp Asp Ser Ala TyrAsp Glu 130 135 140 Pro Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln Tyr GluLeu Gln Tyr 145 150 155 160 Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg ProVal Thr Lys Leu Ile 165 170 175 Ser Val Asp Ser Arg Asn Val Ser Leu LeuPro Glu Glu Phe His Lys 180 185 190 Asp Ser Ser Tyr Gln Leu Gln Val ArgAla Ala Pro Gln Pro Gly Thr 195 200 205 Ser Phe Arg Gly Thr Trp Ser GluTrp Ser Asp Pro Val Ile Phe Gln 210 215 220 Thr Gln Ala Gly Glu Pro GluAla Gly Trp Asp Pro His Met Leu Leu 225 230 235 240 Leu Leu Ala Val LeuIle Ile Val Leu Val Phe Met Gly Leu Lys Ile 245 250 255 His Leu Pro TrpArg Leu Trp Lys Lys Ile Trp Ala Pro Val Pro Thr 260 265 270 Pro Glu SerPhe Phe Gln Pro Leu Tyr Arg Glu His Ser Gly Asn Phe 275 280 285 Lys LysTrp Val Asn Thr Pro Phe Thr Ala Ser Ser Ile Glu Leu Val 290 295 300 ProGln Ser Ser Thr Thr Thr Ser Ala Leu His Leu Ser Leu Tyr Pro 305 310 315320 Ala Lys Glu Lys Lys Phe Pro Gly Leu Pro Gly Leu Glu Glu Gln Leu 325330 335 Glu Cys Asp Gly Met Ser Glu Pro Gly His Trp Cys Ile Ile Pro Leu340 345 350 Ala Ala Gly Gln Ala Val Ser Ala Tyr Ser Glu Glu Arg Asp ArgPro 355 360 365 Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Gly Asp AlaGlu Gly 370 375 380 Leu Cys Val Trp Pro Cys Ser Cys Glu Asp Asp Gly TyrPro Ala Met 385 390 395 400 Asn Leu Asp Ala Gly Arg Glu Ser Gly Pro AsnSer Glu Asp Leu Leu 405 410 415 Leu Val Thr Asp Pro Ala Phe Leu Ser CysGly Cys Val Ser Gly Ser 420 425 430 Gly Leu Arg Leu Gly Gly Ser Pro GlySer Leu Leu Asp Arg Leu Arg 435 440 445 Leu Ser Phe Ala Lys Glu Gly AspTrp Thr Ala Asp Pro Thr Trp Arg 450 455 460 Thr Gly Ser Pro Gly Gly GlySer Glu Ser Glu Ala Gly Ser Pro Pro 465 470 475 480 Gly Leu Asp Met AspThr Phe Asp Ser Gly Phe Ala Gly Ser Asp Cys 485 490 495 Gly Ser Pro ValGlu Thr Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg 500 505 510 Gln Trp ValVal Arg Thr Pro Pro Pro Val Asp Ser Gly Ala Gln Ser 515 520 525 Ser<210> SEQ ID NO 86 <211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC3424 <400> SEQUENCE: 86 aacagctatg accatg 16<210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC694 <400> SEQUENCE: 87 taatacgact cactataggg 20<210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC24399 <400> SEQUENCE: 88 agcggtctca gcctacagtg20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC24400 <400> SEQUENCE: 89 tgagctgggg acaacaaggt20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC24806 <400> SEQUENCE: 90 tgacgaaccc tccaactacg20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Oligonucleotide primer ZC24807 <400> SEQUENCE: 91 tgctctcagc caggacaaag20

What is claimed is:
 1. A method of using a polypeptide to detect anatural ligand from lymphoid cells comprising: isolating a polypeptidecomprising a sequence of amino acid residues selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 237 (His); (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 255 (Leu); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Cys), to amino acidnumber 538 (Ser); and (d) the amino acid sequence as shown in SEQ IDNO:2 from amino acid number 1 (Met) to amino acid number 538 (Ser); andexposing the polypeptide shown in (a) through (e) to activated CD3+selected human T-cell conditioned media, and wherein the polypeptideexhibits cell proliferation activity or signal transduction activitywhen exposed to activated CD3+ selected human T-cell conditioned media;and thereby detecting the natural ligand.
 2. The method of using apolypeptide according to claim 1, further comprising isolating apolypeptide comprising amino acids 20 (Cys), to amino acid number 237(His ) of SEQ ID NO:2 or amino acids 20 (Cys) to amino acid number 538(Ser) of SEQ ID NO:2 to use as an antagonist to competitively inhibitthe cell proliferation or signal transduction activity of the naturalligand from lymphoid cells.
 3. A method of using a polypeptide to detecta natural ligand from lymphoid cells comprising: transfecting a cellwith a vector comprising a transcription promoter; a DNA segmentencoding a polypeptide comprising a sequence of amino acid residuesselected from the group consisting of: (a) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Cys), to amino acidnumber 237 (His); (b) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 255 (Leu); (c) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 538 (Ser); and (d) the amino acid sequenceas shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acidnumber 538 (Ser); and a transcription terminator, wherein the promoteris operably linked to the DNA segment, and the DNA segment is operablylinked to the transcription terminator; and wherein the cell expressesthe polypeptide encoded by the DNA sequence; and exposing the cellexpressing the polypeptide to activated CD3+ selected human T-cellconditioned media, wherein the polypeptide exhibits cell proliferationor signal transduction activity when exposed to activated CD3+selectedhuman T-cell conditioned media; and thereby detecting the naturalligand.
 4. The method of using a polypeptide according to claim 3,further comprising: isolating a polypeptide comprising amino acids 20(Cys), to amino acid number 237 (His ) of SEQ ID NO:2 or amino acids 20(Cys) to amino acid number 538 (Ser) of SEQ ID NO:2 to use as anantagonist to competitively inhibit the cell proliferation or signaltransduction activity of the natural ligand from lymphoid cells.
 5. Themethod of claim 1, wherein the polypeptide consists of a sequence ofamino acid residues selected from the group consisting of: (a) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 20 (Cys),to amino acid number 237 (His); (b) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 20 (Cys), to amino acid number 255(Leu); (c) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 20 (Cys), to amino acid number 538 (Ser); and (d) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) toamino acid number 538 (Ser).
 6. The method of using a polypeptideaccording to claim 5, further comprising: isolating a polypeptidecomprising amino acids 20 (Cys), to amino acid number 237 (His ) of SEQID NO:2 or amino acids 20 (Cys) to amino acid number 538 (Ser) of SEQ IDNO:2 to use as an antagonist to competitively inhibit the cellproliferation or signal transduction activity of the natural liganldfrom lymphoid cells.
 7. The method of claim 3, wherein the DNA segmentencodes a polypeptide consisting of a sequence of amino acid residuesselected from the group consisting of: (a) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Cys), to amino acidnumber 237 (His); (b) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 255 (Leu); (c) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 538 (Ser); and (d) the amino acid sequenceas shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acidnumber 538 (Ser).
 8. A method for detecting a zalpha11 receptor ligandwithin a test sample, comprising: obtaining a test sample comprisinglymphoid cells, hematopoeitic cells, activated T-cells, or cancerouscells, or conditioned medium from lymphoid cells, hematopoeitic cells,activated T-cells, or cancerous cells; contacting the test sample with apolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Cys), to amino acid number 237 (His); (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 255 (Leu); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Cys), to amino acidnumber 538 (Ser); and (d) the amino acid sequence as shown in SEQ IDNO:2 from amino acid number 1 (Met) to amino acid number 538 (Ser); anddetecting the binding of the polypeptide to a ligand in the sample. 9.The method according to claim 8, wherein the polypeptide furthercomprises a transmembrane domain and an intracellular domain.
 10. Themethod according to claim 8, wherein the polypeptide is membrane boundwithin a cultured cell, and the detecting step comprises measuring abiological response in the cultured cell.
 11. The method according toclaim 10, wherein the biological response is cell proliferation, signaltransduction activity, or activation of transcription of a reportergene.
 12. The method according to claim 8, wherein the polypeptide isimmobilized on a solid support.
 13. A method according to claim 8,wherein the polypeptide further comprises a biotin/avidin label,radionuclide, enzyme, substrate, cofactor, inhibitor, fluorescentmarker, chemiluminescent marker, toxin, cytotoxic molecule or animmunoglobulin Fc domain.
 14. A method for detecting a zalpha11 receptorligand within a test sample, comprising: obtaining a test samplecomprising lymphoid cells, hematopoeitic cells, activated T-cells, orcancerous cells, or conditioned medium from lymphoid cells,hematopoeitic cells, activated T-cells, or cancerous cells; contactingthe test sample with a polypeptide consisting of an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Cys), to amino acidnumber 237 (His); (b) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 255 (Leu); (c) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Cys), to amino acid number 538 (Ser); and (d) the amino acid sequenceas shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acidnumber 538 (Ser); and detecting the binding of the polypeptide to aligand in the sample.
 15. The method according to claim 14, wherein thepolypeptide further comprises a transmembrane and an intracellulardomain.
 16. The method according to claim 14, wherein the polypeptide ismembrane bound within a cultured cell, and the detecting step comprisesmeasuring a biological response in the cultured cell.
 17. The methodaccording to claim 16, wherein the biological response is cellproliferation, signal transduction activity, or activation oftranscription of a reporter gene.
 18. The method according to claim 14,wherein the polypeptide is immobilized on a solid support.
 19. Themethod according to claim 14, wherein the polypeptide further comprisesa biotin/avidin label, radionuclide, enzyme, substrate, cofactor,inhibitor, fluorescent marker, chemiluminescent marker, toxin, cytotoxicmolecule or an immunoglobulin Fc domain.
 20. The method according toclaim 14, wherein the polypeptide consists of a sequence of amino acidresidues as shown in SEQ ID NO:2 from amino acid number 20 (Cys), toamino acid number 237 (His), and wherein the polypeptide furthercomprises a transmembrane domain and an intracellular domain from aheterologous cytokine receptor.
 21. The method according to claim 20,wherein the heterologous cytokine receptor is a class I cytokinereceptor.
 22. The method according to claim 14, wherein the polypeptideconsists of a sequence of amino acid residues as shown in SEQ ID NO:2from amino acid number 20 (Cys), to amino acid number 255 (Leu), andwherein the polypeptide further comprises an intracellular domain from aheterologous cytokine receptor.
 23. The method according to claim 22,wherein the heterologous cytokine receptor is a class I cytokinereceptor.